Glucosamine and glucosamine/anti-inflammatory mutual prodrugs, compositions, and methods

ABSTRACT

Mutual prodrugs of glucosamine, and derivatives and analogs of glucosamine and an anti-inflammatory agent, compositions thereof, and methods for, e.g., treating disorders and conditions by administration of the compositions are provided. Topical compositions of glucosamine, and derivatives and analogs of glucosamine are also provided.

This application is a U.S. National Stage Application of InternationalApplication No. PCT/US2005/011739, filed Apr. 7, 2005, which claims thebenefit of U.S. Provisional Application No. 60/560,128, filed Apr. 7,2004, which is incorporated herein by reference in its entirety.

STATEMENT OF GOVERNMENT RIGHTS

This invention was made with Government support under Grant GM066321-01awarded by the National Institutes of Health. The Government may havecertain rights in the invention.

BACKGROUND OF THE INVENTION

An estimated 21 million adults in the United States alone live withosteoarthritis, one of the most common types of arthritis.Osteoarthritis, also called degenerative joint disease, is caused by thebreakdown of cartilage, the connective tissue that cushions the ends ofbones within the joint. Osteoarthritis is characterized by pain, jointdamage, and limited joint motion. This disease generally occurs late ina patient's life, and most commonly affects the hands and largerweight-bearing joints. Additionally, age, gender (females), and obesityare risk factors for this disease.

Researchers have found that in degenerating cartilage, pro-inflammatorycytokines, such as IL-1β and TNFα, are associated with an increaseddegradation of cartilage matrix (Sandy et al., Biochem. J., 335:59-66(1998); Séguin et al., J. Cell Physiol., 197:356-369 (2003)). Theseevents are also correlated with the reduction in the cartilage matrixgene expression and syntheses in vitro (Gouze et al., FEBS Letters,510:166-170 (2002); Shikhman et al., J. Immunol., 166:5155-5160 (2001)).

The amino monosaccharide glucosamine, naturally occurring in cartilageand connective tissues, contributes to maintaining strength,flexibility, and elasticity of these tissues. Glucosamine is a precursorto a glycosaminglycan molecule, which is used in the formation andrepair of cartilage. In vivo, glucosamine is typically converted toN-acetyl glucosamine. In recent years, glucosamine has been used widelyto treat the symptoms of osteoarthritis in human and animal models,serving in an anti-inflammatory capacity in reducing joint swelling andpain levels comparable with that observed with non-steroidalanti-inflammatory drugs (NSAIDs) (Lopes, Curr. Med. Res. Opin.,8:145-149 (1982); Muller-Fassbender et al., Osteoarthritis Cartilage,2:61-69 (1994); Ruane et al., Br. J. Community Nurs., 7:148-152 (2002)).Some have also concluded that glucosamine counteracts the degradativeeffects that IL-1β has on proteoglycan syntheses (Sandy et al., BiochemJ., 335:59-66 (1998); Gouze et al., FEBS Letters, 510:166-170 (2002)),as glucosamine reduces nitric oxide production induced by IL-1β and TNFα(Shikhman et al., J. Immunol., 166:5155-5160 (2001)) and suppresses thesyntheses of cyclooxygenase-2 (COX-2) by human chondrocytes in responseto IL-1β (Largo et al., Osteoarthritis Cartilage, 11:290-298 (2003)).Thus, glucosamine may also serve in the management of diseasesassociated with degeneration of cartilage tissues, such asosteoarthritis.

The use of glucosamine gained popularity after being featured in thebook, The Arthritis Cure by Jason Theodasakis, Md., et al. (St. Martin'sPress. New York, N.Y. 1997). Between 1997 and 2002, the annual marketgrowth rate of glucosamine has exceeded 36.4% (Chemical Market Reporter,vol. 264(1), Jul. 14, 2003). Currently, glucosamine and its metabolitesare not classified as drugs, but as nutraceutical/dietary supplementsunder United States Food and Drug Administration's Dietary SupplementHealth and Education Act of 1994 (DSHEA). Oral dosage formulations ofN-acetyl-D-glucosamine and its parent compound glucosamine in salt form(sulfate, hydrochloride etc.) are commercially available nutraceuticals,and are commonly administered in conjunction with chondroitin sulfate,also a readily available nutraceutical. Glucosamine and chondroitin havebeen reported effective in the oral treatment of osteoarthritis but havenot undergone the rigorous studies needed for FDA approval aspharmaceuticals. (Theodasakis et al., The Arthritis Cure, 1^(st)Edition, St. Martin's Press. New York, N.Y. 1997; McAlindon et al.,JAMA, 283:469-1475 (2000)). The National Institutes of Health (Bethesda,Md., USA) has an ongoing multi-center study-GAIT(Glucosamine/Chondroitin Arthritis Intervention Trial) that is currentlyevaluating the efficacy of orally administered glucosamine andchondroitin oral supplements (Glucosamine/Chondroitin ArthritisIntervention Trial (GAIT), National Center for Complementary andAlternative Medicine (NCCAM), and National Institute of Arthritis andMusculoskeletal and Skin Diseases (NIAMS) September 1999).

While oral administration is the most widely recognized method ofadministering glucosamine, the effectiveness of glucosamine administeredsubcutaneously has also been studied. For example, there currentlyexists an FDA approved therapy, SYNVISC (Genzyme Corp., Naarden, theNetherlands) for the local treatment of pain associated withosteoarthritis of the knee. The treatment includes injection of asolution including sodium hyaluronate (a glycosaminoglycan) at theaffected joint. However, SYNVISC is currently approved only fortreatment of the knee.

Non-steroidal anti-inflammatory drugs (NSAIDs) are effective in reducinginflammation, and are often used to treat the symptoms ofosteoarthritis. However, NSAIDs may have undesirable side effects.Efforts have been made to improve the pharmaceutical properties ofNSAIDs, such as permeability, solubility, and stability, by creatingNSAID “prodrugs.” A prodrug is a drug precursor. The term “prodrug” hasbeen used to describe a compound that is composed of one active drugcompound and a second, non-active compound. The prodrug is not active asa pharmacological agent until it undergoes a chemical conversion, e.g.,via metabolic processes after administration to a patient. Onceconverted, the prodrug provides the active pharmaceutical agent and thenonactive compound that is typically inert after conversion.

The prodrug concept was initially articulated by Albert (Nature,182(4633):421-423 (1958)). The original objectives of prodrug synthesisand development were to improve drug stability and to target drugdelivery for drugs administered orally and intravenously. Stability issignificant to drug activity, and for water and enzyme labile drugs,stability is typically achieved by protecting the drug from chemicalhydrolysis and enzyme degradation subsequent to drug administration.Targeted delivery for prodrugs is based on enhancing drug solubility andpermeability, and is particularly useful in drug administrationassociated with lipid membranes in order to penetrate the veryhydrophobic blood brain barrier.

The most common form of prodrug utilizes an ester linkage formedsynthetically through reaction of a carboxylic acid with an alcohol orphenol to modify the parent drug's in vivo metabolic fate. In additionto affecting the metabolism of the parent drug, the ester prodrug maypossess other advantages, such as reduced side effects. For examplegastric distress may be reduced if the nonsteroidal anti-inflammatorydrug (NSAID) were formulated as a prodrug, as compared with the NSAIDadministered alone. Positive characteristics associated with prodrugusage include, for example, the presence of stable covalent esterlinkage, less intrinsic activity compared to the parent drug, lowertoxicity, and better release kinetics at the binding site to ensureeffective drug levels.

“Mutual prodrugs,” representing a variation of a prodrug, can bedescribed as the conjugation of two drugs having differentpharmacological activities. The concept arises from the practice ofclinically co-administering two drugs in order to enhancepharmacological activity or prevent clinical side effects (U.S. Pat. No.4,278,679). Mutual prodrugs are synthesized toward a pharmacologicalobjective of improving each drug's efficacy, optimizing delivery, andlowering toxicities.

In a mutual prodrug, each component drug functions as the “pro” portionwith respect to the other. Like a prodrug, a mutual prodrug is convertedinto the component active drugs within the body through enzymatic and/ornon-enzymatic reactions. Mutual prodrugs can be classified as, forexample, carrier-linked prodrugs, bio-precursor prodrugs, or chemicalactivation prodrugs, depending upon their constituents and composition(Albert, Nature, 182(4633):421-423 (1958); Rao, H Surya Prakash(available on the internet at ias.ac.in/resonance/Feb2003/pdf/Feb2003p19-27.pdf), Capping Drugs: Development of Prodrugs.k February, 2003). Atthe site of action, the side effects of the original drug would bemasked allowing the drug to work more effectively (Albert, Nature,182(4633):421-423 (1958)). Mutual prodrugs are typically similar tosingle active agent prodrugs in regard to pharmaceutical andpharmacological activities, such as absorption, disposition, metabolism,and excretion. The objective of a mutual prodrug is for both activedrugs reaching their respective active sites, to provide the desiredpharmacological effects while minimizing adverse metabolic and/ortoxicological events.

For many years before the terms “prodrug” and “mutual prodrug” werecoined in the research domain, combination drugs have been administeredto patients as therapeutic agents (Singh et al., Indian J. Pharm. Sci,56(3):69-79 (1994)), for example in relation to the production ofsulphasalazine, representing modern advances in antibiotic prodrugs.Essentially, this has led to combinations of β-lactam antibiotics andtheir potentiating agents to produce, for example ampicillin-mecillinamand ampicillan-sulbactam to form sultamicillin, and Dual ActionCephalosporins as well as other agents not typically referred to asmutual prodrugs (Singh et al., Indian J. Pharm. Sci, 56(3):69-79 (1994))

One example of a mutual prodrug is estramustine sodium phosphate (EMCYT,Pharmacia, La Roche) developed in the early 1970's as an anti-neoplasticagent that shows certain mutual prodrug characteristics (Wang et al.,Biochem. Pharmacol., 55(9):1427-33 (1998); Sheridan et al., CancerSurv., 11:239-254 (1991); Ohsawa et al., Gan To Kagaku Ryoho., April;15(4 Pt 2-1):1065-71 (1998); Forsgren et al., Urol Nephrol Suppl.,107:56-58 (1988)). Estramustine is typically used in the treatment ofmetastatic carcinoma of the prostate. Estramustine is selectively takenup into estrogen receptor positive cells and then, as shown in FIG. 1,the urethane linkage is hydrolyzed to give 17-alphaestradiol, whichslows prostate cell growth, and nornitrogen mustard as a weak alkylatingagent.

Prodrug research has continued, as exemplified by the synthesis of5-fluorouracil/cytarabine mutual prodrugs designed to reduce theresistance mechanisms at work in the delivery of single nucleoside drugs(Menger et al., J. Org. Chem., 62:9083-9088 (1997)). Researchers such asBhosale and co-workers have made attempts to produce mutual prodrugs ofibuprofen/paracetamol and ibuprofen/salicylaminde. The goal of this workwas to produce prodrugs of NSAIDS to reduce the associated side effects(Bhosale et al., Indian J. Pharm. Sci., 66(2):158-163 (2003)). Theirapproach was unique from the perspective of producing mutual prodrugsvis-à-vis physicochemical modifications towards simplistic NSAIDdelivery, much like sulphasalazine, currently used more so as aulcerative colitis therapeutic consisting of sulphapyridine and5-aminosaicylic acid covalently bound via an azo bond (Klotz et al.,Adv. Drug Deliv. Rev., 57(2):267-279 (2005); Lim et al., Rev.Gastroenterol. Disord., 4(3):104-117 (2004); Baker et al., Rev.Gastroenterol. Disord., 4(2):86-91 (2004); and Diculescu et al., Rom. J.Gastroenterol., 12(4):283-286 (2003). Structures of Sulphasalazine (FIG.2A), 5-Fluorouracil/Cytarabine (FIG. 2B), Ibuprofen/Paracetamol (FIG.2C), and Ibuprofen/Salicylamide (FIG. 2D) are provided in FIG. 2.

SUMMARY OF THE INVENTION

The concept of a “mutual prodrug” is relatively new in medicinalchemistry, pharmaceutics, and drug delivery. A mutual prodrug iscomposed of two drug compounds that are covalently linked, for example,by an ester linkage (Ueda et al., Mem. Inst. Sci Ind. Res. Osaka Univ.,47:43-54 (1990); Imai et al., J. Pharmacol. Exp. Ther., 265:328-333(1994); Fukuhara et al., Chirality, 8:494-502 (1996); Fukuhara et al.,Biol. Pharm. Bull., 18:140-147 (1995); and Otagiri et al., J. Con.Release, 62:223-229 (1999)). When covalently linked, the drug componentsare rendered pharmaceutically inactive; however, the linkage providessome beneficial aspect to the mutual prodrug, such as improved deliveryof the covalently linked drugs, as compared with delivery of each of thedrugs individually. An ester linkage is easily degraded by mammalianesterase, thereby allowing release of each drug component in vivo. Eachof the drug components is thereby rendered pharmaceutically active.Thus, after administration to a patient, cleavage of the mutual prodrugpermits each of the drug components, pharmaceutically activated bycleavage, to produce its respective intended pharmacological action. Ina mutual prodrug, each component facilitates the delivery of the othercomponent.

In one aspect, the present invention provides a compound that functionsas a mutual prodrug. The compound includes two pharmaceutically activesubstances. In particular, the present invention is directed to acompound including a first component covalently linked to a secondcomponent, said compound having formula I:

wherein R¹, R², R³ and R⁴ are each independently H or an organic group;L is an optional linking group; and X is an anti-inflammatory agent. Ina preferred embodiment, R¹, R², R³ and R⁴ are each H; L is an acetylgroup; and X is ibuprofen or ketoprofen.

The first component, represented by the substituted ring structurelinked to X, the anti-inflammatory agent, through the linker L, ispreferably glucosamine or a derivative or analog of glucosamine. Thesecond component, X, is an anti-inflammatory agent, preferably anonsteroidal anti-inflammatory agent (NSAID). The linker L may bepresent or absent in the compound having formula I. If linker L ispresent, the first component is considered to be indirectly linked tothe second component. If linker L is absent, the first component isconsidered to be directly linked to the second component. The linkagebetween the first and second components, whether direct or indirect, isa covalent linkage.

The linkage between the first and second component is a cleavablelinkage. For example, the linkage may be hydrolyzable and/or may beenzymatically cleavable. Preferably, the linkage is cleavable underphysiological conditions, such as those present in a mammalian body,particularly a human body. When a linker L is used, the linkage betweenthe first component and L is cleavable, and/or the linkage between L andthe second component, X, is cleavable.

In addition to facilitating delivery of the active components, thelinking of the components may impart a protective effect on the mutualprodrug, thereby reducing or preventing unwanted degradation, usually bystomach acids, and/or side effects of either or both of the drugs, priorto cleavage of the mutual prodrug in vivo. This protective effectafforded by the mutual prodrug may be particularly desirable if an NSAIDis one of the drugs, in view of the side effects common to these drugs.The mutual prodrugs of the present invention may also exhibitaqueous/lipid solubility profiles different than those of each drugindividually, which may further aid in improvements in formulationand/or delivery of the drugs.

The invention further provides a pharmaceutical composition thatincludes a compound having formula I. In one embodiment of theinvention, a composition is provided that includes the compound havingformula I, as described above, and a pharmaceutically acceptablecarrier. The composition is preferably formulated for topicalapplication.

In another aspect, the present invention provides a pharmaceuticalcomposition that includes a therapeutically effective amount of acompound having formula II:

wherein R¹, R², R³, R⁴, and R⁵ are each independently H or an organicgroup; and a pharmaceutically acceptable carrier; wherein thecomposition is formulated for topical application. In a preferredembodiment, R¹, R², R³ and R⁴ are each H and R⁵ is an acetyl group. Thecompound of formula II is preferably glucosamine or a derivative oranalog of glucosamine. While glucosamine and derivatives thereof aretypically delivered orally, as, for example, nutraceuticals, and, lesscommonly, delivered subcutaneously, the present invention advantageouslyprovides for the topical delivery of glucosamine and glucosaminederivatives, or their acetylated analogs.

Optionally, topical and/or transdermal application of a glucosamine, ora derivative or analog thereof, can be accompanied by co-administrationof an anti-inflammatory agent, such as an NSAID. When administeredtogether, the anti-inflammatory agent and the glucosamine, includingderivatives and analogs thereof, may, but need not, be covalently linkedto form a mutual prodrug, as described in greater detail herein.

In another aspect, the present invention provides a method for treatingand/or preventing a disorder or a condition in a mammal that includesadministering to the mammal a therapeutically effective amount of acomposition of the invention. The method can involve a therapeutic,prophylactic and/or cosmetic use. In one embodiment, the invention isdirected to a method to alleviate a condition treatable with glucosaminewhich method includes administering to a mammal, preferably a human, aneffective amount of a compound having formula I or II as describedherein. In another embodiment, the invention is directed to a method toalleviate a condition treatable with an anti-inflammatory agent whichmethod includes administering to a mammal an effective amount of acompound having formula I or II as described herein.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a diagram of the structure of estramustine sodium phosphate(EMCYT) and its promoieties.

FIGS. 2A-2D show structures of certain prodrug models. FIG. 2A shows adiagram of the structure of the prodrug sulphasalazine; FIG. 2B shows adiagram of the structure of projected mutual prodrug model5-fluorouracil/cytarabine; FIG. 2C shows a diagram of the structure ofprojected mutual prodrug model ibuprofen/paracetamol; and FIG. 2D showsa diagram of the structure of projected mutual prodrug modelibuprofen/salicylamide.

FIGS. 3A and 3B show structures of indomethacin and glucamethacin,respectively.

FIG. 4 shows an exemplary scheme for synthesis of a spacer linked mutualprodrug of the invention.

FIG. 5 shows a scheme for synthesis of a directly linked mutual prodrugof the invention.

FIG. 6 shows a scheme for synthesis of a spacer linked mutual prodrugthat includes a glycosaminoglycan and an NSAID.

FIG. 7 shows a scheme for synthesis of a directly linked mutual prodrugthat includes a glycosaminoglycan and an NSAID.

FIG. 8 shows a differential scanning calorimetry (DSC) thermograph ofthe mutual prodrug synthesized according to the scheme of FIG. 4(compound 6).

FIG. 9 shows an expanded DSC thermograph of the mutual prodrugsynthesized according to the scheme of FIG. 4 (compound 6).

FIG. 10 shows a DSC thermograph of the mutual prodrug synthesizedaccording to the scheme of FIG. 5 (compound 9).

FIG. 11 shows a DSC thermograph of the mutual prodrug synthesizedaccording to the scheme of FIG. 5 (compound 9) showing sublimation,phase change, and degradation phenomena of the compound.

FIG. 12 shows a graph of time vs. peak area as a function ofconcentration for a diffusion study of compositions of (a) glucosamineHCl, (b) N-acetyl glucosamine, and (c) glucosamine pentaacetyl.

FIG. 13 is a graph of the effect of DMSO on the cumulative permeation ofN-acetyl glucosamine through shed snake skin.

FIG. 14 is a bar graph showing the accumulation of N-acetyl glucosaminethrough shed snake skin from phosphate buffer (pH 5.5) including 2%, 5%,10%, 25%, and 50% ethanol, by percentage volume of aqueous phase andethanol containing NAG.

FIG. 15 is a graph showing the effect of ethanol concentration oncumulative permeation of N-acetyl glucosamine at 37.5° C. through shedsnake skin at 2%, 5%, 10%, 25%, and 50% ethanol, by percentage volume ofaqueous phase and ethanol containing NAG.

FIG. 16 is a graph showing the effect of soy lecithin-vitamin E onN-acetyl glucosamine permeation across shed snake skin.

FIG. 17 is a graph showing physicochemical data obtained from thepermeation of N-acetyl glucosamine in pluronic gel-organic phasevehicles through shed snake skin.

DETAILED DESCRIPTION OF ILLUSTRATIVE EMBODIMENTS

Mammals, both humans and animals, commonly suffer from certain diseasesassociated with the deterioration of cartilage and joint connectivetissue, such as osteoarthritis, which can cause pain, swelling,stiffness, and limited mobility of the affected joint. Management of thesymptoms has typically been carried out through treatment withanti-inflammatory agents, such as NSAIDs. Anti-inflammatory agents arewell documented for their pharmacological, toxicological, andbiopharmaceutical properties in the treatment of pain and inflammationassociated with diseases such as osteoarthritis.

The administration of NSAIDs may, however, cause certain deleteriousside effects in some patients. For example, NSAIDs may cause stomachupset, stomach ulcers and/or intestinal bleeding. Also, persons havingcertain conditions, such as thyroid disease, diabetes, heart disease,high blood pressure, and allergies, as well as persons who are about tohave surgery (including dental surgery) and pregnant women may sufferserious side effects from administration of NSAIDs. Further,anti-inflammatory drugs do not treat an underlying cause of thedisorder, e.g., deterioration of cartilage and connective tissues.

Glucosamine is currently being investigated as a possible treatment fordiseases characterized by degeneration of e.g., cartilage and connectivetissues. There is evidence that treatment with glucosamine not onlyeases the pain of, e.g., osteoarthritis, but it may also treat thedisease itself, as it has been shown to slow the progression ofdegeneration and to re-grow cartilage tissue. Glucosamine is thusexpected to be effective in the treatment of osteoarthritis, although itis currently not regulated in the United States as an activepharmaceutical ingredient (API).

Glucosamine is typically administered orally; however it is consideredto have poor bioavailability as an orally administered nutraceutical.Only a small percentage of the active ingredient (e.g., 12-13%) isbelieved to be available to the affected tissue following oraladministration.

Subcutaneous injection of a glucosamine has also been investigated. Forexample, SYNVISC, a device for administering a hyaluronic acidderivative available from Genzyme Corp. (Naarden, the Netherlands), is atherapy that is FDA approved only for the local treatment of painassociated with osteoarthritis of the knee. SYNVASC is oftenadministered to patients “off-label,” that is, it is administered toother (e.g., non-approved) joints, thus providing anecdotal evidencethat glucosamine is effective as a treatment for osteoarthritis.Injection of a drug, however, carries its own potential problems, e.g.,injection site infection, patient aversion to injection, difficulty ofadministration, etc. Thus, improved dosage forms, providing greaterbioavailability of glucosamine, its derivatives and analogs, and moreeffective methods of its delivery to the affected areas of a patient areneeded.

The present invention provides for effective delivery of glucosamine,including derivatives and analogs thereof, optionally delivered in theform of a mutual prodrug with an anti-inflammatory agent, to jointsaffected with diseases associated with degeneration of cartilage andconnective tissue.

As used herein, the term “glucosamine” is understood to refer toglucosamine, derivatives of glucosamine, analogs of glucosamine, andmetabolites of glucosamine, unless otherwise indicated.

As used herein, the term “organic group” is understood to mean ahydrocarbon group (with optional elements other than carbon andhydrogen, such as oxygen, nitrogen, sulfur, and silicon) that isclassified as an aliphatic group, cyclic group, or combination ofaliphatic and cyclic groups (e.g., alkaryl and aralkyl groups).

The term “aliphatic group” means a saturated or unsaturated linear orbranched hydrocarbon group. This term is used to encompass alkyl,alkenyl, and alkynyl groups, for example. The term “alkyl group” means asaturated linear or branched hydrocarbon group including, for example,methyl, ethyl, isopropyl, t-butyl, heptyl, dodecyl, octadecyl, amyl,2-ethylhexyl, and the like. The term “alkenyl group” means anunsaturated, linear or branched hydrocarbon group with one or morecarbon-carbon double bonds, such as a vinyl group. The term “alkynylgroup” means an unsaturated, linear or branched hydrocarbon group withone or more carbon-carbon triple bonds. The term “cyclic group” means aclosed ring hydrocarbon group that is classified as an alicyclic group,aromatic group, or heterocyclic group. The term “alicyclic group” meansa cyclic hydrocarbon group having properties resembling those ofaliphatic groups. The term “aromatic group” or “aryl group” means amono- or polynuclear aromatic hydrocarbon group. The term “heterocyclicgroup” means a closed ring hydrocarbon in which one or more of the atomsin the ring is an element other than carbon (e.g., nitrogen, oxygen,sulfur, etc.). A group that may be the same a or different from anothergroup is referred to as being “independently” something.

The present invention provides a novel compound that is a mutual prodrugcombining a first component, glucosamine, or a derivative or analog ofglucosamine, and a second component, an anti-inflammatory agent. Thepresent invention further provides a pharmaceutical compositionincluding the mutual prodrug compound to facilitate delivery of theactive agents. Once the mutual prodrug delivered via the composition andis cleaved in vivo, the first component and the second component arerendered active and mutually provide the therapeutic benefits of NSAIDsand glucosamine and its analogs and derivatives, such asglycosaminoglycans. Pain may be managed, for example, via COX-1/COX-2inhibition mechanisms of a non-steroidal anti-inflammatory agentdelivered as a component of the mutual prodrug, with the added benefitthat, as the anti-inflammatory is delivered topically, substantially allpotential side effects, particularly gastrointestinal side effects, arereduced or eliminated. The maintenance and/or repair of tissues viaregulation of cellular events and/or physiological processes, such ascell-cell and cell-matrix interactions, and cellproliferation/differentiation may be managed by, e.g., aglycosaminoglycan or ester of glycosaminoglycan as the other componentof the mutual prodrug.

Additionally, while not wishing to be held to any particular theory, theanti-inflammatory component of the mutual prodrug may assist in thepercutaneous delivery of the glucosamine, thus increasing thebioavailability of the glucosamine upon cleavage of the mutual prodrugin vivo. Additionally, the anti-inflammatory itself also providestreatment of the affected areas.

The mutual prodrug compound of the present invention is represented byformula I:

wherein R¹, R², R³ and R⁴ are each independently H or an organic group,as defined above, X is an anti-inflammatory agent, and L is an optionallinking group. If no linking group, L, is present, the anti-inflammatoryagent, X, is directly linked to the first component. Linking groups areof any type such that the first component (to the left of linker L informula I) is covalently linked to the second component, X (to the rightof linker L in formula I), and wherein the linking group is cleavable,either between the N(H)-L bond, the L—X bond, and/or internally withinthe linker L, in such a manner in vivo as to render the first componentand the second component active. Typically the linking group is anorganic group, as defined above. Preferred linking groups include anacyloxy ester and an alpha hydroxyl ester.

The first component of the mutual prodrug compound provides theglucosamine portion of the mutual prodrug. While any glucosamine of thestructure indicated above is suitable for use in the mutual prodrugcompounds of the present invention, certain glucosamines may providepreferred embodiments. Such glucosamines include, but are not limitedto, for example, glucosamine, glucosamine pentaacetate,glucosamine-1-phosphate, glucosamine-6-phosphate,N-acetyl-β-D-glucosamine, N-acetylglucosamine-6-phosphate,N-acetylglucosamine-1-phosphate, uridine diphosphate-N-acetylglucosamine, 2-amino-2-deoxy-1,3,4,6-acetyl-β-D-glucopyranose, theacetylated analog of 2-amino-2-deoxy-1,3,4,6-acetyl-β-D-glucopyranose,2-acetamido-2-deoxy-β-D-glucopyranose-1,3,4,6-tetraacetate, theacetylated analog of2-acetamido-2-deoxy-β-D-glucopyranose-1,3,4,6-tetraacetate, andN-acetylglucosamine (NAG). Particularly preferred embodiments of themutual prodrug of the present invention includeamino-2-deoxy-1,3,4,6-acetyl-β-D-glucopyranose,2-acetamido-2-deoxy-β-D-glucopyranose-1,3,4,6-tetraacetate, andN-acetylglucosamine (NAG).

As indicated above, the mutual prodrugs of the present invention includeas a second component an anti-inflammatory agent covalently attached tothe glucosamine component, as described in more detail below.

The components of the mutual prodrug typically remain inactive untilthey are converted to their active forms in vivo by breaking thecovalent bond between the components. The anti-inflammatory agent may beattached directly to the glucosamine (e.g., replacing one of the aminohydrogens), wherein upon cleavage in vivo, only the two active speciesof the mutual prodrug (the anti-inflammatory and the glucosamine) areprovided. Alternatively, the anti-inflammatory agent may be covalentlyattached to the glucoamine via a linking group or spacer, L, forexample, an ester group. Upon delivery of this embodiment of the mutualprodrug, the bonds between the first and/or second components and thelinking group are cleaved, e.g., by esterases present in vivo, themutual prodrug providing the two active species (the anti-inflammatoryand the glucosamine). The linking groups, if present, are preferablyselected such that the entire linking group is cleaved from theglucosamine and the anti-inflammatory, whereupon the linking group isreleased and optionally degraded or otherwise metabolized.

Any type of anti-inflammatory agent capable of covalently bonding to theglucosamine, as described above, and which provides desired effects to apatient are suitable for use in the mutual prodrugs of the presentinvention. Such anti-inflammatory agents include, for example,prostaglandins, arachidonic acid, metabolites of arachidonic acid,non-steroidal anti-inflammatory agents and derivatives of non-steroidalanti-inflammatory agents.

Non-steroidal anti-inflammatory agents (NSAIDs) are particularly usefulin view of their wide availability, their effectiveness as ananti-inflammatory, and, for some NSAIDs, their relatively low cost.Preferred NSAIDs useful in the mutual prodrug compounds of the presentinvention include, but are not limited to, for example, salicylic acids(e.g., acetylsalicylic acid (aspirin), choline magnesium trisalicylate,diflunisal, salsalate, magnesium salicylate, choline salicylate, cholinemagnesium salicylate, sodium salicylate), propionic acids (e.g.,fenoprofen, fenoprofen calcium, flurbiprofen, ibuprofen, ketoprofen,naproxen, oxaprozin, pirprofen, indobufen, indoprofen, tiaprofenicacid), acetic acids (e.g., diclofenac, indomethacin, glucametacin,sulindac, tolmetin, carprofen) enolic acids (e.g., meloxicam, piroxicam,tenoxicam, lornoxicam) fenamic acids (e.g., meclofenamate, meclofenamatesodium, mefenamic acid, etofenamat), napthylalkanones (e.g.,nabumetone), pyranocarboxylic acids (e.g., etodolac), pyrroles (e.g.,ketorolac, ketorolac tromethamine, phenylbutazone, remifenzone),para-aminphenols (e.g., acetaminophen), and cyclooxygenase-2 (COX-2)inhibitors (e.g., celecoxib, valdecoxib, rofecoxib, flosulide,nimesulide). The structures of indomethacin and glucametacin are shownin FIGS. 3A and 3B respectively. Preferred NSAIDs for use in the mutualprodrug of the present invention include ibuprofen and ketoprofen. Inone embodiment, the compound indomethacin is excluded from the group ofNSAIDs used in the mutual prodrug of the invention.

The invention additionally provides pharmaceutical compositions. Thecompositions of the present invention may include the mutual prodrugcompound of formula I as described above in a pharmaceuticallyacceptable carrier, as described in more detail below.

The invention also provides a composition that includes a glucosamine,or a derivative or analog of glucosamine (e.g., a compound havingformula II), in a formulation for topical and/or transdermal delivery.These topical glucosamine compositions are believed to providepercutaneous transport/permeability across skin membrane such thatbioavailability of glucosamine, and consequently its effectiveness, ascompared with the glucosamine bioavailability of oral glucosamineformulations, may be improved.

Additional compositions of the present invention may include aglucosamine (e.g., the compound of formula I) in a pharmaceuticallyacceptable carrier, wherein the composition is, preferably, formulatedfor topical and/or transdermal delivery. These compositions do notinclude the mutual prodrug; however, formulations as disclosed hereinhave been shown to provide unexpected percutaneous transport across skinmembrane, which, it is anticipated, may increase the bioavailability ofthe glucosamine.

As used herein and unless otherwise indicated, topical administration isfunctionally the same as transdermal administration. While not wishingto be held to any particular theory, topical administration ofglucosamine may provide a greater bioavailability of the activeingredient. Up to the present, however, it has proven difficult toachieve permeation across skin membranes of glucosamines. The presentinvention provides pharmaceutical compositions including glucosaminesthat have improved permeability, thus providing a greaterbioavailability of the active agent.

Certain useful compositions of the invention include, but are notlimited to, e.g., esters of glycosaminoglycans, such as chrondroitin,dermatan, heparin, heparin, keratin, and other biologically significantproteoglycans. Examples of certain useful glycosaminoglycan esters,which are components of connective tissue and cartilage, include2-amino-1,3,4,6-acetyl-beta-D-glucopyranosyl;2-acetamido-2-deoxy-beta-D-glucopyranose-1,3,4,6-tetraacetate; and2-acetamido-2-deoxy-beta-D-glucopyranose.

Compositions of the present invention including one or more glucosaminesare provided by, for example, formulating or admixing the glucosamine(s)in a pharmaceutically acceptable carrier, for example a cream, gel,solution, ointment, lotion, suspension, emulsion, micoremulsion,liposome, transdermal patch, etc. Such cream, gel, solution, ointment,lotion, suspension, emulsion, mocroemulsion, liposome, or transdermalpatch may include any number or combination of topical/transdermalvehicles approved by the United States Pharmacopoeia (USP) for human orveterinary use, for example, fatty esters, alcohols, gel bases (e.g.,pluronic gels) lecithin, dimethylsulfoxide (DMSO), water, etc. to form atransdermal and/or topical agent. If either a subcutaneous or an oralformulation is desired, the pharmaceutically acceptable carrier mayinclude, for example, normal saline, pluronic F-127 solution, or agenerally recognized as safe (GRAS) delivery solution (subcutaneous,intramuscular, and/or intravenous delivery), or tablets, capsules,powders, suspensions, emulsions, and/or gels (oral delivery).

The pharmaceutical compositions of the present invention may include oneor more additional components appropriate for use in the composition andthat, when included, will provide the desired results (e.g., willprovide additional therapeutic benefits to skin or tissues, or will aid,for example, in the consistency and stability of the composition).Additional components may include, for example, antimicrobial agents,gelling agents, emulsifying agents, stiffening agents, skin healingagents, emollients surfactants, solvents, lubricants, waxes, humectants,skin penetration enhancers, anti-oxidants, and any combination of these.

While it is understood that certain components useful in thecompositions of the present invention may provide more than one effectin a composition depending upon, e.g., concentration of the component(for example, a poloxamer may be considered both a gelling agent and anemulsifier), the following are typical additional components useful inthe present compositions.

Anti-microbial agents are typically present in a composition to assistin preserving the composition, thus extending its shelf life.Anti-microbial agents useful in the present invention include ethanol,parabens, salts of parabens, sorbic acid, potassium sorbate, propyleneglycol, glycerin, etc. Combinations of these anti-microbial agents mayalso be used.

Certain additional components may be useful in enhancing penetration ofthe composition through the skin membranes. Useful skin penetrationenhancers include, for example, dimethyl sulfoxide, ethanol,polyethylene glycol, urea, dimethyl acetamide, sodium lauryl sulfate,Spans, Tweens, terpenes, azone, acetone, and oleic acid. A preferredskin penetration enhancer, particularly for e.g., veterinary use, isdimethyl sulfoxide (DMSO). A preferred skin penetration enhancer,particularly for use in administration to human patients, is ethanol.

While the present compositions may themselves provide treatment to skinfor such conditions as skin damage, burns, and age-related wrinking ofthe skin, in addition to providing therapeutic effects to such diseasesas osteoarthritis, it may be desired to include additional skin healingand/or anti-oxidant providing components for additional therapeuticbenefit to the skin. Such skin healing agents include, for example,vitamin E, vitamin E-tocopherol polyethylene glycol succinate (vitaminE-TPGS), ascorbic acid, alpha tocopherol, beta tocopherol, gammatocopherol, aloe vera, etc. A particularly useful skin healing agent inthe present compositions is vitamin E. Additionally, certain usefulanti-oxidants include fumaric acid, malic acid, ascorbic acid palmitate,butylated hydroxylanisole, propyl gallate, sodium ascorbate, sodiummetabisulfite, etc.

Certain other additional components, such as emulsifying agents andstiffening agents, may be used to provide, e.g., stability and desiredconsistency to the compositions. Typical emulsifying agents useful inthe present invention include cholesterol, poloxamers, lecithin,carbomers, polyoxyethylene ethers, fatty acid esters, stearates, etc.Certain preferred emulsifying agents include poloxamers and lecithin,with a particularly preferred poloxamer being Pluronic F-127.Additionally, typical stiffening agents useful in the presentcompositions include long chain fatty alcohols and long chain fattyalcohol esters.

In addition to any of the above additional components, one or more ofthe anti-inflammatory agents discussed above in connection with themutual prodrug of the present invention may be included as a freecomponent (e.g., not covalently bonded to the glucosamine or in additionto a composition including the mutual prodrug) in any of thecompositions of the present invention. Furthermore, as theseanti-inflammatory agents are not covalently linked to the glucosamine,choice of anti-inflammatory present as a free agent is not thuslylimited.

To provide pharmaceutically effective compositions, the first componentof the compositions of the present invention (e.g., the glucosamine,derivatives thereof and/or analogs thereof) are typically present in thecomposition in an amount of at least about 1 percent by weight, andpreferably at least about 10 percent by weight. Further, the glucosaminecomponent is typically present in the compositions in an amount of nogreater than about 75 percent by weight, and preferably no greater thanabout 40 percent by weight.

Two general methods for synthesizing mutual prodrugs of the inventionare shown in FIGS. 4 and 5. It should be understood, however, that theinvention is not limited by any particular synthetic method, and othermethods of synthesis are included. Each exemplary method (FIGS. 4 and 5)provides a mutual prodrug that is a glucosamine or derivative thereof,such as a glycosaminoglycan or ester of glycosaminoglycan, coupled withan anti-inflammatory agent, such as an NSAID, and the exemplary mutualprodrug is hydrolysable or otherwise cleavable. Preferably, the linkageis hydrolysable or cleavable in vivo.

Scheme 1 (FIG. 4) is a diagram describing the general synthesis of anexemplary mutual prodrug of the present invention wherein theglucosamine is linked with the anti-inflammatory agent via a spacer(e.g., a linker or linking group), such as an imide-ester covalent boundalkyl chain, covalently bound to the NSAID via the inherently createdmalonic ester configuration. The linking moieties are typically cleavedin vivo to release the glucosamine and the anti-inflammatory agent, aswell as the inert linking group.

Alternatively, mutual prodrugs of the present invention may besynthesized to provide a direct link between the glucosamine, orderivative thereof, and the anti-inflammatory agent according to theexemplary scheme of FIG. 5. Compositions including mutual prodrugssynthesized according to the general scheme of FIG. 5 deliver mutualprodrugs that are preferably cleaved in vivo to provide the glucosamineand the anti-inflammatory without release of an inert component.

The present invention also provides methods of treating a disorder in amammal that includes administration of the compositions of the presentinvention.

The compounds, compositions, and methods of the present invention aresuitable for treating various disorders or conditions in mammals (e.g.,for human and/or veterinary administration), wherein treatment includesrelief from a symptom of the disorder (e.g., pain and/or swelling of ajoint), alleviating a cause of the disorder (e.g., repair ofdeteriorating cartilage and/or connective tissues) and/or improvement ofa condition (e.g., lessening of the appearance of facial wrinkles).

The disorder may be the result of disease (e.g., osteoarthritis) or maybe the result of a physical injury (e.g., joint and muscle sprains).Furthermore, the compositions and methods of the present invention arenot necessarily limited to treatment of disease and/or injury.Compositions of the present invention may provide therapeutic andcosmetic effects to damaged and wrinkled skin by e.g., application ofthe compositions to facial wrinkles and/or other areas of skin damage(e.g., skin damage caused by sun, salt, and/or wind exposure), toprovide soothing of the skin (a therapeutic effect) and at leasttemporary lessening of the appearance of wrinkles in the skin (acosmetic effect).

Thus, disorders and conditions that may be treated according to themethods of the present invention, as discussed herein, include, but arenot limited to, arthritis, osteoarthritis, osteoporosis, muscle sprains,muscle strains, joint sprains, joint strains, tendonitis, bursitis,burns, joint pain, inflamed joints, skin damage, skin tenderness, skinpain, sun-damaged skin, wind-damaged skin, salt-damaged skin, scartissue, age-related wrinkling of the skin, and any combinations thereof.

Methods of treatment according to the present invention includeadministration of a pharmaceutical composition of the invention to apatient using any convenient route, without limitation. In a preferredembodiment, the mutual prodrug is administered to a patient topicallyand/or transdermally, via local application to skin or to other externalor internal membrane, as described more fully above. In otherformulations, the mutual prodrug, or its metabolic products, aredelivered to the bloodstream and circulated systemically.

In another embodiment of the invention, the mutual prodrug isadministered orally, such as in the form of tablets, powders, capsules,suspensions, emulsions, gels, etc. In further embodiments, treatment ofa disorder is achieved via subcutaneous, intramuscular, and intravenousinjection, as more fully described above.

While it is understood that the compositions of the present inventionmay be administered to a patient to treat a number of disorders and/orconditions, which can include, without limitation, therapeutic disordersand/or cosmetic applications, it is also contemplated that manydifferent additional components can be included to provide formulationssuitable for delivery of a composition of the invention with respect tothe disorder or condition of interest.

For example, compositions of the present invention that include aglucosamine, derivative thereof, or analog thereof (e.g., thecompositions that do not include the mutual prodrug) may be preparedfrom various combinations of additional components, e.g., isopropylpalmitate, isopropyl myristate, a glycosaminoglycan or glycosaminoglycanester, a stiffening agent like long chain fatty alcohols, long chainfatty alcohol esters, waxes like spermaceti, nonionicgelling/emulsifiers like poloxamers, water, and a USP approvedantimicrobial agent, wherein the additional components are selected toprovide desired characteristics to the compositions. Further, certain ofthese exemplary formulations may provide enhanced permeability, thusimproved bioavailablilty, of the glycosaminoglycan ester.

Exemplary formulations include, but are not limited to, the following,wherein an NSAID may, optionally, be included in any of theformulations:

1. Isopropyl palmitate, DMSO, ethanol, lecithin, poloxamer, ester ofglycosaminoglycan

2. Isopropyl palmitate, water, ethanol, lecithin, poloxamer, ester ofglycosaminoglycan

3. Isopropyl palmitate, vitamin E, lecithin, poloxamer, ester ofglycosaminoglycan

4. Isopropyl myristate, poloxamer, water, ethanol, lecithin, ester ofglycosaminoglycan

5. Isopropyl myristate, water, lecithin, poloxamer, ester ofglycosaminoglycan

6. Isopropyl palmitate, water, lecithin, poloxamer, USP approvedantimicrobial agent, ester of glycosaminoglycan

7. Vitamin E, lecithin, isopropyl myristate, ester of glycosaminoglycan

8. Isopropyl myristate, lecithin, poloxamer, ester of glycosaminoglycan

9. Isopropyl palmitate, USP approved antimicrobial agent, vitamin E,lecithin, hydrous or anhydrous lanolin, ester of glycosaminoglycan

10. Isopropyl palmitate, USP approved antimicrobial agent, vitamin E,hydrous or anhydrous lanolin, ester of glycosaminoglycan

11. Isopropyl palmitate, USP approved antimicrobial agent, vitamin E,lecithin, ester of glycosaminoglycan

12. Isopropyl palmitate, lecithin, USP approved antimicrobial agent,hydrous or anhydrous lanolin, ester of glycosaminoglycan

13. Isopropyl myristate, vitamin E, USP approved antimicrobial agent,lecithin, hydrous or anhydrous lanolin, ester of glycosaminoglycan

14. Isopropyl myristate, USP approved antimicrobial agent, vitamin E,lecithin, ester of glycosaminoglycan

15. Isopropyl myristate, USP approved antimicrobial agent, vitamin E,hydrous or anhydrous lanolin, ester of glycosaminoglycan

16. Isopropyl myristate, USP approved antimicrobial agent, lecithin,hydrous or anhydrous lanolin, ester of glycosaminoglycan

17. Isopropyl myristate, USP approved antimicrobial agent, vitamin E,ester of glycosaminoglycan

18. Isopropyl myristate, USP approved antimicrobial agent, lecithin,ester of glycosaminoglycan

19. Isopropyl myristate, USP approved antimicrobial agent, hydrous oranhydrous lanolin, ester of glycosaminoglycan

20. Isopropyl myristate, DMSO, ethanol, lecithin, poloxamer, ester ofglycosaminoglycan

21. Isopropyl myristate, vitamin E, Lecithin, hydrous or anhydrouslanolin, ester of glycosaminoglycan

22. Isopropyl myristate, DMSO, ethanol, vitamin E, poloxamer, ester ofglycosaminoglycan

23. Isopropyl myristate, DMSO, vitamin E, poloxamer, ester ofglycosaminoglycan

24. Isopropyl myristate, DMSO, ethanol, vitamin E, poloxamer, ester ofglycosaminoglycan

25. Isopropyl palmitate, DMSO, ethanol, lecithin, vitamin E, poloxamer,ester of glycosaminoglycan

26. Isopropyl palmitate, ethanol, lecithin, poloxamer, ester ofglycosaminoglycan

27. Isopropyl palmitate, DMSO, ethanol, lecithin, ester ofglycosaminoglycan

28. Isopropyl palmitate, DMSO, ethanol, poloxamer, ester ofglycosaminoglycan

29. Isopropyl palmitate, ethanol, lecithin, ester of glycosaminoglycan

30. Isopropyl palmitate, DMSO, ethanol, lecithin, vitamin E, poloxamer,ester of glycosaminoglycan

31. Isopropyl palmitate, isopropyl myristate, DMSO, ethanol, lecithin,poloxamer, ester of glycosaminoglycan

32. Isopropyl palmitate, isopropyl myristate, DMSO, ethanol, lecithin,vitamin E, poloxamer, ester of glycosaminoglycan

33. Isopropyl palmitate, isopropyl myristate, DMSO, ethanol, poloxamer,ester of glycosaminoglycan

34. Isopropyl palmitate, isopropyl myristate, DMSO, ethanol, vitamin E,poloxamer, ester of glycosaminoglycan

35. Isopropyl palmitate, isopropyl myristate, DMSO, ethanol, lecithin,ester of glycosaminoglycan

36. Isopropyl palmitate, isopropyl myristate, DMSO, ethanol, lecithin,vitamin E, poloxamer, ester of glycosaminoglycan

37. Isopropyl palmitate, isopropyl myristate, DMSO, lecithin, vitamin E,poloxamer, ester of glycosaminoglycan

38. Isopropyl palmitate, isopropyl myristate, DMSO, vitamin E,poloxamer, ester of glycosaminoglycan

39. Isopropyl palmitate, isopropyl myristate, DMSO, lecithin, poloxamer,ester of glycosaminoglycan

40. Isopropyl palmitate, isopropyl myristate, DMSO, lecithin, vitamin E,ester of glycosaminoglycan

41. Isopropyl palmitate, isopropyl myristate, DMSO, poloxamer, ester ofglycosaminoglycan

42. Isopropyl palmitate, isopropyl myristate, DMSO, lecithin, ester ofglycosaminoglycan

43. Isopropyl palmitate, isopropyl myristate, DMSO, vitamin E, ester ofglycosaminoglycan

44. Isopropyl palmitate, isopropyl myristate, water, ethanol, lecithin,ester of glycosaminoglycan

45. Isopropyl palmitate, isopropyl myristate, water, ethanol, vitamin E,poloxamer, ester of glycosaminoglycan

46. Isopropyl palmitate, isopropyl myristate, water, ethanol, vitamin E,poloxamer, hydrous or anhydrous lanolin, ester of glycosaminoglycan

47. Isopropyl palmitate, isopropyl myristate, water, ethanol, poloxamer,hydrous or anhydrous lanolin, ester of glycosaminoglycan

48. Isopropyl palmitate, isopropyl myristate, water, ethanol, vitamin E,hydrous or anhydrous lanolin, ester of glycosaminoglycan

49. Isopropyl palmitate, isopropyl myristate, water, ethanol, vitamin E,lecithin, poloxamer, hydrous or anhydrous lanolin, ester ofglycosaminoglycan

50. Isopropyl palmitate, isopropyl myristate, water, ethanol, lecithin,hydrous or anhydrous lanolin, ester of glycosaminoglycan

51. Isopropyl palmitate, isopropyl myristate, water, ethanol, vitamin E,hydrous or anhydrous lanolin, ester of glycosaminoglycan

52. Isopropyl palmitate, isopropyl myristate, water, ethanol, poloxamer,hydrous or anhydrous lanolin, ester of glycosaminoglycan

53. Isopropyl palmitate, isopropyl myristate, water, ethanol, hydrous oranhydrous lanolin, ester of glycosaminoglycan

54. Isopropyl palmitate, isopropyl myristate, water, ethanol, poloxamer,ester of glycosaminoglycan

55. Isopropyl palmitate, isopropyl myristate, water, ethanol, ester ofglycosaminoglycan

56. Isopropyl palmitate, isopropyl myristate, water, USP approvedantimicrobial agent, lecithin, vitamin E, poloxamer, ester ofglycosaminoglycan

57. Isopropyl palmitate, isopropyl myristate, water, USP approvedantimicrobial agent, vitamin E, poloxamer, ester of glycosaminoglycan

58. Isopropyl palmitate, isopropyl myristate, water, USP approvedantimicrobial agent, lecithin, poloxamer, ester of glycosaminoglycan

59. Isopropyl palmitate, isopropyl myristate, water, USP approvedantimicrobial agent, lecithin, vitamin E, ester of glycosaminoglycan

60. Isopropyl palmitate, isopropyl myristate, water, USP approvedantimicrobial agent, lecithin, ester of glycosaminoglycan

61. Isopropyl palmitate, isopropyl myristate, water, USP approvedantimicrobial agent, poloxamer, ester of glycosaminoglycan

62. Isopropyl palmitate, isopropyl myristate, water, USP approvedantimicrobial agent, vitamin E, ester of glycosaminoglycan

63. Isopropyl palmitate, isopropyl myristate, water, ethanol, lecithin,vitamin E, poloxamer, ester of glycosaminoglycan

64. Isopropyl palmitate, isopropyl myristate, ethanol, lecithin,poloxamer, ester of glycosaminoglycan

65. Isopropyl palmitate, isopropyl myristate, DMSO, ethanol, lecithin,ester of glycosaminoglycan

66. Isopropyl palmitate, isopropyl myristate, ethanol, lecithin, vitaminE, poloxamer, ester of glycosaminoglycan

67. Isopropyl palmitate, isopropyl myristate, water, ethanol, lecithin,poloxamer, ester of glycosaminoglycan

68. Isopropyl palmitate, isopropyl myristate, water, USP approvedantimicrobial agent, lecithin, poloxamer, ester of glycosaminoglycan

69. Isopropyl palmitate, isopropyl myristate, DMSO, ethanol, poloxamer,ester of glycosaminoglycan

70. Isopropyl palmitate, isopropyl myristate, ethanol, lecithin, esterof glycosaminoglycan

71. Isopropyl palmitate, isopropyl myristate, USP approved antimicrobialagent, vitamin E, lecithin, hydrous or anhydrous lanolin, ester ofglycosaminoglycan

72. Isopropyl palmitate, isopropyl myristate, USP approved antimicrobialagent, lecithin, hydrous or anhydrous lanolin, ester ofglycosaminoglycan

73. Isopropyl palmitate, isopropyl myristate, USP approved antimicrobialagent, vitamin E, hydrous or anhydrous lanolin, ester ofglycosaminoglycan

74. Isopropyl palmitate, isopropyl myristate, USP approved antimicrobialagent, vitamin E, lecithin, ester of glycosaminoglycan

75. Isopropyl palmitate, isopropyl myristate, USP approved antimicrobialagent, vitamin E, ester of glycosaminoglycan

76. Isopropyl palmitate, isopropyl myristate, USP approved antimicrobialagent, lecithin, ester of glycosaminoglycan

77. Isopropyl palmitate, isopropyl myristate, USP approved antimicrobialagent, hydrous or anhydrous lanolin, ester of glycosaminoglycan

78. Isopropyl palmitate, isopropyl myristate, USP approved antimicrobialagent, vitamin E, lecithin, hydrous or anhydrous lanolin, poloxamer,ester of glycosaminoglycan

79. Isopropyl palmitate, isopropyl myristate, USP approved antimicrobialagent, lecithin, hydrous or anhydrous lanolin, poloxamer, ester ofglycosaminoglycan

80. Isopropyl palmitate, isopropyl myristate, USP approved antimicrobialagent, vitamin E, hydrous or anhydrous lanolin, poloxamer, ester ofglycosaminoglycan

81. Isopropyl palmitate, isopropyl myristate, USP approved antimicrobialagent, vitamin E, lecithin, poloxamer, ester of glycosaminoglycan

82. Isopropyl palmitate, isopropyl myristate, USP approved antimicrobialagent, hydrous or anhydrous lanolin, poloxamer, ester ofglycosaminoglycan

83. Isopropyl palmitate, isopropyl myristate, USP approved antimicrobialagent, lecithin, poloxamer, ester of glycosaminoglycan

84. Isopropyl palmitate, isopropyl myristate, USP approved antimicrobialagent, vitamin E, poloxamer, ester of glycosaminoglycan

85. Isopropyl palmitate, isopropyl myristate, USP approved antimicrobialagent, poloxamer, ester of glycosaminoglycan

86. Isopropyl palmitate, isopropyl myristate, water, USP approvedantimicrobial agent, poloxamer, hydrous or anhydrous lanolin, ester ofglycosaminoglycan

87. Isopropyl palmitate, isopropyl myristate, DMSO, vitamin E, lecithin,hydrous or anhydrous lanolin, poloxamer, ester of glycosaminoglycan

88. Isopropyl palmitate, isopropyl myristate, DMSO, lecithin, hydrous oranhydrous lanolin, poloxamer, ester of glycosaminoglycan

89. Isopropyl palmitate, isopropyl myristate, DMSO, vitamin E, hydrousor anhydrous lanolin, poloxamer, ester of glycosaminoglycan

90. Isopropyl palmitate, isopropyl myristate, DMSO, vitamin E, lecithin,hydrous or anhydrous lanolin, ester of glycosaminoglycan

91. Isopropyl palmitate, isopropyl myristate, DMSO, vitamin E, hydrousor anhydrous lanolin, ester of glycosaminoglycan

92. Isopropyl palmitate, isopropyl myristate, DMSO, lecithin, hydrous oranhydrous lanolin, ester of glycosaminoglycan

93. Isopropyl myristate, oleic acid, ethanol, lecithin, hydrous oranhydrous lanolin, ester of glycosaminoglycan

94. Isopropyl myristate, oleic acid, lecithin, hydrous or anhydrouslanolin, ester of glycosaminoglycan

95. Isopropyl myristate, lecithin, hydrous or anhydrous lanolin, esterof glycosaminoglycan

96. Isopropyl myristate, oleic acid, ethanol, DMSO, vitamin E, lecithin,hydrous or anhydrous lanolin, poloxamer, ester of glycosaminoglycan

97. Isopropyl myristate, oleic acid, DMSO, vitamin E, lecithin, hydrousor anhydrous lanolin, poloxamer, ester of glycosaminoglycan

98. Isopropyl myristate, oleic acid, ethanol, hydrous or anhydrouslanolin, ester of glycosaminoglycan

99. Isopropyl myristate, oleic acid, hydrous or anhydrous lanolin, esterof glycosaminoglycan

100. Isopropyl myristate, oleic acid, ethanol, vitamin E, poloxamer,ester of glycosaminoglycan

101. Fatty esters, ester of glycosaminoglycan

102. Isopropyl palmitate, isopropyl myristate, glycosaminoglycan orglycosaminoglycan ester, cetyl alcohol, poloxamer, water, antimicrobialagent

103. Isopropyl palmitate, isopropyl myristate, glycosaminoglycan orglycosaminoglycan ester, cetyl alcohol, antimicrobial agent

104. Isopropyl palmitate, isopropyl myristate, glycosaminoglycan orglycosaminoglycan ester, stearyl alcohol, poloxamer, water,antimicrobial agent

105. Isopropyl palmitate, isopropyl myristate, glycosaminoglycan orglycosaminoglycan ester, stearyl alcohol, antimicrobial agent

106. Isopropyl palmitate, isopropyl myristate, glycosaminoglycan orglycosaminoglycan ester, cetostearyl alcohol, poloxamer, water,antimicrobial agent

107. Isopropyl palmitate, isopropyl myristate, glycosaminoglycan orglycosaminoglycan ester, cetostearyl alcohol, antimicrobial agent

108. Isopropyl palmitate, isopropyl myristate, glycosaminoglycan orglycosaminoglycan ester, spermaceti (or spermaceti replacement),poloxamer, water, antimicrobial agent

109. Isopropyl palmitate, isopropyl myristate, glycosaminoglycan orglycosaminoglycan ester, spermaceti (or spermaceti replacement),antimicrobial agent

110. Isopropyl palmitate, isopropyl myristate, glycosaminoglycan orglycosaminoglycan ester, cetyl esters, poloxamer, water, antimicrobialagent

111. Isopropyl palmitate, isopropyl myristate, glycosaminoglycan orglycosaminoglycan ester, cetyl esters, antimicrobial agent

112. Isopropyl palmitate, isopropyl myristate, glycosaminoglycan orglycosaminoglycan ester, emulsifying wax, poloxamer, water,antimicrobial agent

113. Isopropyl palmitate, isopropyl myristate, glycosaminoglycan orglycosaminoglycan ester, emulsifying wax, antimicrobial agent

Certain of the above formulations of the invention will naturally bebetter suited for certain purposes. For example, one formulation toenhance percutaneous absorption of the composition at a joint, such asthe knee, will likely be different from a formulation intended toheal/restore sun damaged skin around the eyes and/or wrinkles around theeyes caused by exposure to sun and weather. The latter use benefits fromthe inclusion of vitamin E to promote skin healing. However, vitamin Emay not provide any added benefit to a formulation intended forpenetration of the skin and into the joint, even though vitamin E wouldnot interfere with the skin penetration process.

Thus cosmetic formulations, for example, are exemplified by formulationentries 1-101, while formulations suited for use in treating arthriticand/or inflamed joints are exemplified, for example, by formulationentries 102-113. It should be understood that, regardless of theintended use of the composition, the compositions and methods of thepresent invention are not limited by the selection of the components inthe listed formulations 1-113.

EXAMPLES

The present invention is illustrated by the following examples which aredirected to the mutual prodrugs and to the topical and/or transdermalapplication of glucosamines, including derivatives and esters thereof,as described above. It is to be understood that the particular examples,materials, amounts, and procedures set forth below are to be interpretedbroadly in accordance with the scope and spirit of the invention as setforth herein. Further, the examples are provided for clarity ofunderstanding, and the invention is not limited to the exact details asshown and described; many variations will be apparent to one skilled inthe art.

Example 1 NSAID-Glucosamide Mutual Prodrug Development and Synthesis

Combining NSAIDs and glucosamine into a single mutual prodrug allowsdelivery of these drugs concomitantly in a form that can targetdisorders such as osteoarthritis by either oral or transdermaladministration. Transdermal delivery is advantageous because it avoidsside effects associated with oral delivery of NSAIDs, such as adversedrug reactions and/or adverse gastrointestinal effects, oftenexperienced with oral administration to patients of NSAIDs (Shi et al.,Acta Pharmacol. Sin., 25(3), 357-365 (2004); Benini et al., Pediatr.Nephrol., 19(2):232-234 (2004); Wiholm, Curr. Med. Res. Opin.,17(3):210-216 (2001); Kromann-Andersen et al., Dan. Med. Bull.,35(2):187-192 (1988); Pietzsch et al., Int. J. Clin. Pharmacol. Ther.,40(3):111-115 (2002); Karch et al., JAMA, 22, 234(12):1236-1241 (1975))and also the metabolism/excretion events often observed withadministration of glucosamine (Setnikar et al., Arzneimittel-Forschung,36(4):729-735 (1986); Aghazadeh-Habashi et al., Journal of Pharmacy &Pharmaceutical Sci., 5(2):181-184 (2000); Setnikar et al,Arzneimittel-Forschung, 43(10): 1109-13 (1993); and Setnikar et al.,Arzneimittel-Forschung, 51(9):699-725 (2001)).

Experimental

An objective of the present study was to synthesize directly-linked andchained-linked NSAID-glucosamidemutual prodrug models.

Materials and Methods

All reagents and solvents utilized were purchased from FisherScientific. TLC and preparative TLC chromatographs were performed onAnaltech Co. UNIPLATES. Melting points were determined on a Fisher-Johnsapparatus and are uncorrected. Nuclear magnetic resonance spectra wererecorded on Varian INOVA 500 MHz spectrometer for ¹H NMR and ¹³C NMRwith tetramethysilane as an internal stand. Chemical shifts (δ) arereported in parts per million (ppm) and signals are reported as s(singlet), d (doublet), t (triplet), m (multiplet), or br (broadsinglet). A Beckman DU-650 and a Thermo Electron Corp. AQUAMATE wereused to record the UV spectra. Staff at the University of Georgia'sChemical and Biological Sciences Mass Spectrometry Facility completedESI (electrospray ionization) mass spectra. Column chromatographs wereperformed using silica gel>440 mesh. Differential Scanning Calorimetry(DSC) was performed on a Perkin-Elmer DSC 7 with TAC 7/DX utilizingPYRIS Thermal Analysis System (Rev. E/March 2002) software.

Synthesis

Synthesis of the mutual prodrug models were successfully completed toproduce compound products 6 (Scheme I, FIG. 6) and 9 (Scheme II, FIG.7). Methods were explored that would preserve the carbohydrate'sβ-conformation, protect the hydroxy (OH) groups to allow the amine (NH₂)to undergo selective addition to produce primary intermediates 4 (SchemeI, FIG. 6) and 7 (Scheme II, FIG. 7). Compounds 1-3 (Scheme I, FIG. 6)were synthesized from procedures adapted from: Bergman et al., Chem.Ber., 1932, 975; Silva et. Al., J. Org. Chem., 64:5926-5929 (1999)(Supplemental Material); and Chauviere et al., J. Med. Chem., 46:427-220(2003) as starting materials towards compound 6.

Scheme I (FIG. 6)

Preparation of 1,3,4,6-tetra-O-acetyl-2-deoxy-2-{[{[2-(4-isobutylphenyl)propanoyl]oxy}(phenyl)acetyl]amino}-β-D-glucopyranose (Compound 6)

2-deoxy-2-amino-1,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl (4) Compound 3(100 g, 0.26 mol) was titrated with triethylamine to yield a whiteprecipitate. The precipitate was filtered and washed with CH₂Cl₂ (2×150milliliters (ml)). The filtrate was dried under vacuum for 24 hours. Theorganic layer was washed with brine (2×100) and dried with Mg₂SO₄. Thesolvent was removed via reduced pressure rotary evaporation and productdried for 24 hours under vacuum to provide Compound 4, afforded as awhite solid (87.9 grams (g), 97% yield). ¹H NMR (d-acetone) 9.21 s, 1H),6.15 (d, 1H), 5.38 (t, 1H), 5.08 (t, 1H), 4.31 (dd, 1H), 4.11-4.03 (m,2H), 3.56 (t, 1H), 3.03-2.05 (dd, 6H), 2.25 (d, 3H), 2.10-2.09 (m, 3H).¹³C NMR (d-acetone) 205.7, 170.1, 169.87, 169.39, 169.0, 95.20, 74.85,72.28, 68.61, 61.90, 55.46, 19.98, 19.85, 19.82, 19.77. ES1 forC14H21NO9: FW 347 found in m/z 348 [M+H+]. Mp 134° C.

2-deoxy-2-(2-chloro-2-phenyl)acetylamino-1,3,4,6-tetra-O-acetyl-β-D-glucopyranosyl(5)

Alpha-Chlorophenylacetyl chloride (20 g, 0.105 mol) was added drop-wiseto stirring solution of Compound 4 (29.88 g, 0.105 mol), triethylamine(12.4 ml, 0.90 ml) in 50 ml CH₂Cl₂ at −10° to room temperature for 24hours. The reaction mixture was washed with HCl (1.5 N, 2×7 ml), H₂O(1×100 ml) and brine (1×100 ml). The organic phase was dried with Mg₂SO₄and the solvent removed via reduced pressure rotary evaporation. Theresultant syrup was crystallized with ice-cold acetonitrile and driedunder vacuum for 24 hours to yield Compound 5 (27.4 g, 93.5%) affordedas a white solid. ¹H NMR (d-acetone) 7.80 (s, 1H), 7.37 (s, 2H), 7.25(s, 2H), 5.79 (s, 1H), 5.33 (d, 2H), 4.90 (s, 1H), 4.09 (d, 2H), 3.95(s, 1H), 3.84 (s, 1H), 3.17 (s, 1H) 1.87-1.64 (m, 12H). ¹³C NMR(d-acetone) 205.55, 16.88, 169.69, 169.18, 168.51, 167.69, 128.85,128.61 (2C), 127.81 (2C) 91.03, 68.54, 61.70, 60.60 53.13, 19.73, 19.70(2C), 19.62. ES1 for C14H21NO9: FW [M+H+] 499 found m/z 500 440 [M+H+].Mp>200(238)° C.

1,3,4,6-tetra-O-acetyl-2-deoxy-2-{[{[2-(4-isobutylphenyl)propanoyl]oxy}(phenyl)acetyl]amino}-β-D-glucopyranose (6)

Compound 5 (653 milligrams (mg), 1.45 mmol) andα-methyl-4-[isobutyl]phenylacetic acid-Na salt in anhydrous 10 ml CH₂Cl₂was stirred at room temperature for 16 hours. The solution was washedwith brine (2×10 ml) and reduced via rotary evaporation to give Compound6 (763 mg, 93.5%) as a white powder. ¹H NMR (d-acetone) 7.73 (s, 1H),7.41-7.13 (m, 9H), 5.81 (d, 2H), 5.38 (m, 1H), 5.04 (m, 1H), 4.26 (s,2H), 4.10 (s, 1H), 3.96 (s, 2H), 2.48 (s, 2H), 2.01 (s, 6H), 1.84 (s,6H), 1.65 (s, 1H), 1.53 (s, 3H), 0.90 (s, 6H). ¹³C NMR (d-acetone)205.69, 173.37, 169.61, 168.73, 168.70, 140.38, 138.43, 138.03, 129.25,128.36, 127.31, 126.84, 91.96, 75.91, 72.54, 71.66, 68.31, 52.07, 44.61,21.72. 19.61, 17.96: Uv 203λ nm. ES1 for C₃₅H₄₃NO₁₂ FW 669 found m/z 522[M+H+] w/loss of (C₁₁H₁₅).

Scheme II (FIG. 7)

Preparation of 2-deoxy-2-[2-(3-benzoylphenyl)propanoicacid]amino-β-D-glucopyranosyl (Compound 9)2-deoxy-2-amino-1,3,4,6-tetra-O-triethylsilyl-β-D-glucopyranosyl (7)

Glucosamine HCl (7.32 g, 33.95 mmol) and a catalytic amount ofdimethylaminopyridine (DMAP, cat.) was stirred in 100 ml anhydrouspyridine for three hours at room temperature. Chlorotriethylsilane(20.47 g, 136.27 millimoles (mmol)) was added drop wise while thesolution stirred on an ice bath at −5° C. to room temperature for 16 hrsand at 40-45° C. for 2 hours. The pyridine was removed and the resultingoil was washed with 250 ml of ethyl acetate and subsequently with 250 mlof 1:1 ethyl acetate and brine. The final organic layer was dried withNa₂SO₄ and solvent removed via rotary evaporation to give a colorlessoil, which was dried overnight under vacuum, affording Compound 7 (21.82g; 97%) as a white foam. ¹H NMR (CD3OD): 5.39 (d, 1H), 483-4.80 (m, 2H),3.89-3.47 (m, 3H), 3.09-3.01 (dd, 1H), 1.04-0.53 (m, 60H). ES1 forC₃₀H₆₉NO₅Si₄: FW 636 found m/z 636 [M+] and 522 [M+H+] w/loss of(C₆H₁₅).

2-deoxy-2-[2-(3-benzoylphenyl)propanoic acid]amino-β-D-glucopyranosyl(9)

Compound 7 (28 g, 43.5 mmol) and DMAP (cat.) was stirred in 10 mlacetonitrile on an ice bath. In a separate vessel,(2S)-2-(3-benzoylphenyl)propanoic acid (11 g, 43.55 mmol) in 10 mlacetonitrile was stirred on an ice bath with BuNMe₂ (8.75 ml, 120 mmol),Me₂NSO₂Cl (9.67 ml, 90 mmol) until the solution became clear. Bothsolutions were mixed and stirred at 0° C. to room temperature over 12hours. The acetonitrile was removed via rotary evaporation. Theresulting syrup was washed with 2×150 water/ethyl acetate (1:1) and theorganic layer washed with 2×100 ml brine. The organic layer was removedvia rotary evaporation and the resulting syrup dried in vacuo for 12hours to give 2-deoxy-2-(2-(3-benzoylphenyl) propanoicacid)amino-1,3,4,6-tetra-O-triethylsilyl-β-D-glucopyranosyl (Compound 8)(43 g), quantitative: ES1 for C₄₆H₈₁NO₇Si₄: FW 872 found m/z 857 [M+H+]w/loss of CH₃) as a yellow syrup. Compound 8 was directly de-protectedwith t-butyl ammonium fluoride/MeOH to give white precipitate that wasfiltered, washed with methanol and recrystallized from hot ethanol togive Compound 9 (17.3 g, 85%) as an opaque solid. ¹H NMR (DMSO-d6)7.63-7.24 (m, 9H), 6.17 (s, 1H), 4.65 (s, 1H), 4.56 (s, 1H), 4.44 (s,1H), 4.21 (s, 1H), 3.61 (s, 1H), 3.45-2.10 (m, 4H), 2.83 (d, 2H), 2.25(d, 3H), 1.1 (s, 3H). ¹³C NMR (DMSO-d6) 197.13, 176.39, 141.67, 137.64,137.34, 132.54, 131.73, 129.69 (2C), 128.79, 128.47, 128.41 (2C),128.19, 95.20, 74.85, 72.28, 68.61, 61.90, 55.46, 45.09, 18.12. ES1 forC₂₂H₂₅NO₇: FW 416 found m/z 416 [M+H+]. Mp 164° C.

Results

Compounds 6 and 9 are projected to be mutual prodrugs to treat disorderssuch as osteoarthritis via the delivery of both a NSAID and aglucosamine (or ester or derivative thereof).

Compound 6 is ibuprofen covalently bound via a linker to the amide ofglucosamine, and compound 9 is a ketoprofen molecule directly linked toglucosamine, each a model MP. Physiological, enzymatic, and hydrolysisreactions are expected to affect each mutual prodrug's ester andimido-ester linkage respectively, thus making them ideal mutual prodrugsused to target the associated pain and perhaps root cause of disorderssuch as osteoarthritis by either oral or transdermal administration.

Though glucosamine is not currently recognized as a pharmaceutical inthe United States, studies have shown that orally administeredglucosamine promotes glycosaminoglycan synthesis and the production ofproteoglycans that compose the lubricating fluids and support jointtissues (i.e. cartilage) thus, for example, treating osteoarthritis'root cause (McClain et al., Diabetes, 45:1003-1009 (1996); Singh et al.,Diabetes, 50:2355-2362 (2001)). Glucose and glucosamine are substratesof glucokinase (Singh et al., Diabetes, 50:2355-2362 (2001)).Phosphorylated glucosamine, glucosamine-6-phosphate inhibits glucokinaseand alters both glucose and subsequent glucosamine metabolism (VanSchaftigen et al., Biochem. J., 308:23-29 (1995); Virkamaki et al.,Diabetes, 48:1101-1107 (1999)). Miwa et. al reported that glucokinasehas a low affinity for NAG. Thus, NAG kinase mediates thephosphorylation of NAG to produce NAG-6-phosphate that does not affectglucokinase activity (Miwa et al., Enzyme Protein, 48:135-142 (1994)).Concluding that NAG-6-phosphate does not affect glucokinase activitythus allowing glucose and glucosamine to proceed through metabolismunrestricted. The biosynthesis of glycosaminoglycans from thisperspective would be better promoted with the use of NAG or some otherrate-limiting glucosamine analogue rather than by parent glucosamine(Gouze et al., FEBS Lett., 510:166-170 (2002)). Anastassiades, et alreports that glucosamine and analogues thereof such as NAG as well asglucosamine with varying N-linkage-chains have shown degrees of humanchondrocyte cell culture growth via matrix matrix gene expression invitro (Poustie et al., Pharmacol. Exp. Ther., 311(2):610-616 (2004)).From the pharmaceutics perspective per review and taking considerationof Anastassiades patents and patent applications (United States PatentApplication Nos. 20040152665 (2003) and 20020045597 (2001);International Patent Application Publication No. WO 2002017890 A2); ourassumed hypothesis states that by protecting the glucosamine's amide,the half-life of the glucosamine molecule is increased which affects itsactivity. Chain linkage effects have been shown in numerous literaturestudies such as coupling a polymer to a molecule via an ester bond toincrease its half-life as method to modify a chemical entity'sdissolution properties and/or biopharmaceutical properties (D'Souza etal., Journal of Pharmaceutical Sciences, 93(8):1962-1979 (2004)).

A hindrance to the usefulness of glucosamine as a treatment for certaindisorders is its bioavailability, which is typically accepted to beapproximately 12-13%, although some studies seem to indicate someeffective potency in mild to moderate cases of osteoarthritis (Setnikaret al., Arzneimittel-Forschung, 36(4):729-35 (1986); Biopharmaceutics &Drug Disposition, 25(3):109-116 (2004); Du et al., Biopharmaceutics &Drug Disposition, 25(3):109-116 (2004)). Although not intending to beheld to any particular theory, it is our belief that these twosynthesized mutual prodrugs will undergo in vivo hydrolysis to give theparent compounds. Then, each parent compound may provide itstherapeutically recognized effect. Studies have not been completed todetermine whether of the new mutual prodrugs dissolution profile isfaster than their hydrolysis or enzymatic degradation rate constants.Although this is expected, the rates are predicted to vary since thelinkages depend on the chemical nature of the covalent bonds, structureof the compounds and the surrounding conditions in vivo/in vitro.

Compound 6, the tripartite entity may undergo hydrolysis more easilythan the bipartite compound 9. The ibuprofen molecule of compound 6 hasthe potential to undergo hydrolysis due to the linking group. Thelinking group's benzyl moiety could possibly then undergo anintramolecular reaction and/or enzymatic reaction that release theglucosamine molecule. Compound 9, the bipartite compound is expected tohydrolyze to the parent compounds ketoprofen and glucosamine.

These predictions however have been made based on the “in silico”calculated pKa values of compounds 6 and 9 (FIGS. 6 and 7,respectively). The in silico pKa and log P calculations of compound 6were obtained as a result of ACD/pKa v8.02 using the ACD/I-Lab onlineservice (available on the internet at acdlabs.com) and are as follows:

-   -   Ionic form: HL    -   pKa₁ (HL/H+L; 9)=10.79±0.70    -   pKa₂ (H2L/H+HL; 9)=−3.67±0.70    -   Calculated log P: 6.45±0.82

The in silico pKa and log P calculations of compound 9 were alsoobtained as a result of ACD/pKa v8.02 using the ACD/1-Lab online serviceand are as follows:

-   -   Ionic form: H5L    -   pKa₁ (HL/H+L; 9)=15.53±0.70    -   pKa₂ (H2L/H+HL; 11)=14.69±0.70    -   pKa₃ (H3L/H+L; 11)=14.33±0.70    -   pKa₄ (H4L/H+HL; 10)=13.53±0.70    -   pKa₅ (H5L/H+L; 8)=12.03±0.70    -   pKa₆ (H6L/H+HL; 9)=−2.40±0.70    -   Calculated log P: 3.81±0.48

Predictive calculations may provide an estimation of thebiopharmaceutical properties. The pKa calculations reported are derivedfrom algorithms derived from known pKa's or various chemical groups.Here we mainly focus on the pKa of the amide and/or ester linkages. Thenegative pKa of the amide in compound 6 suggests a large Ka value, whichimplies that the equilibrium constant lies to the right for thedissociation of the imido-ester bond via a hydrolysis type cleavagelending itself more to enzymatic cleavage, which can also be the casewith the imido-ester of compound 6. On the other hand, the amide alsohas a high pKa value and, depending on its ionization, the imide-esterbond may be hydrolyzed. Furthermore, the pKa values near 2.5-3.0 aretypically expected to be due to the protonation of the negativelycharged oxygen. Our predictions show this to be possible on the imidoester's carbonyl oxygen. Additional studies will determine the truepathway of whether or not these mutual prodrugs have substratespecificity, hydrophilicity and/or other influences that affect therelease rates of the parent drugs. Development based on theirphysicochemical properties and understanding of their mechanisms ofrelease are the primary determining factors towards further mutualprodrug development.

The ‘in silico’ log P predictions of the compound 6 and 9 (FIGS. 6 and7, respectively) are 6.45±0.82 and 3.81±0.48 respectively. Currently,using Lipinski's Rule of 5 (Du et al., Biopharmaceutics & DrugDisposition, 25(3):109-116 (2004)), compounds 6 and 9 rate a number of 4and 2 respectively. Whereas, these numbers are solubility ranking basedon a collection of chemical compounds estimated from data mining, e.g.using the “rule of five” to determine “drug likeness.” The “rule of 5”states that poor absorption or permeation is more likely when:

-   -   A) There are more than 5H-bond donors (expressed as the sum of        OHs and NHs);    -   B) The MWT is over 500;    -   C) The LogP is over 5 (or MLogP is over 4.15); and    -   D) There are more than 10H-bond acceptors (expressed as the sum        of Ns and Os).

Poor absorption or permeability is possible since neither compoundsatisfies more than two criteria. Though more drug likeness is indicatedfor compound 9 than compound 6 in regard to their aqueous solubility andintestinal permeability based on the “rule of 5.” In light of this,mutual prodrugs containing carbohydrate moieties, compounds such asthese potentially fall into classes that are substrates for biologicaltransporters, which are exceptions to the rule.

This study was primarily commenced as a pharmaceutics study tosynthesize and evaluate the physicochemical properties of two proposedmutual prodrug candidates. Though the dissolution characteristics havenot been discussed, we found the differential scanning calorimetry (DSC)data to be intriguing. In order to evaluate these mutual prodrugs'dissolution characteristics, a consistent crystal form must beestablished, which we failed to achieve. We encountered commonlyobserved carbohydrate phase transitions such as glassy state dynamics,which follow trends of the Tool-Narayanaswany-Moynihan model (Andreozziet al., Journal of Physics: Condensed Matter, 15(11):S1215-S1226(2003)). This model is an attempt to explain the glassy statetransitions observed with carbohydrates and other chemical entities suchas sucrose, trehalose, and (poly)vinylpryrolidone. Its application iswidely used in the pharmaceutical industry to investigate thephysicochemical stability and the quantitative relationship between thewidth of the glass transition and fragility/activation energy forstructural relaxation (Pikal et al., J. of Pharm. Sci., 93(4):981-994(2004)).

In FIG. 8, the DSC thermograph of compound 6, a powder, shows threetransitions indicative of polymorphs that look to be thermally stable,with phase transitions at 226.93° C., 233.09° C. and 237.52° C.respectively. All thermographs were consistent with heating at variousrates. After opening all of the DSC pans, we found that the compound hadsublimed and decomposed.

The DSC thermographs of compound 9 (FIGS. 10 and 11) were difficult toobtain. Compound 9's uncorrected melting point was determined to be 164°C. Representative DSC thermograph of compounds 6 and 9 show exothermicactivity (FIGS. 9 and 11), and many phase transition points indicativeof sublimation, which was proven upon visual inspection. The uncorrectedmelting point is actually the decomposition point in DSC thermograph.Visually, compound 6 was obtained as a syrup, which was recrystallizedunder high vacuum pressure and/or solvent recrystallization as opaquecrystals with elliptical surfaces. To obtain a clearer, picture ofcompound 6, we tried x-ray crystallography, which failed due to theopaqueness and complexity of the crystal structure(s). No distinctpolymorph and glassy transition states were observed. Pikal et al.'shypothesis, “ . . . because physical and chemical degradation processesrequire atomic and molecular mobility, just as structural relaxationrequires similar mobility, instability processes are correlated or‘coupled’ to structural relation” (J. Pharm. Sci., 93(4):981-994 (2004))seems to be valid regarding compound 6 as an amorphous chemical entity.

The two mutual prodrug models were synthesized to follow the sameprocesses and/or methodologies as prodrugs in attempts to overcomepharmaceutical and pharmacological problems such as incompleteabsorption, too rapid absorption and excretion observed with NSAIDS andglucosamine. The end objective was to have an NSAID and glucosaminereach a site providing its pharmacological effect while minimizing theadverse drug events and/or effects. It is anticipated that in vitroand/or in vivo studies will verify the anticipated objective of thesenew synthesized mutual prodrugs, as well as derivatives and/or analogsthereof.

We have also developed and performed preliminary performulation studiesof other glucosamine-NSAID analogs as a delivery system for glucosamineand NSAIDs to produce anti-inflammatory effect as well as cartilagegrowth. The NSAID can be, for example, ibuprofen or ketoprofen.

Example 2 Solubility and Transport of Glucosamine-NSAID Mutual Prodrug

Transdermal deliver of an NSAID-glycosamineglycan mutual prodrug, suchas ibuprofen-glucosamine, can concomitantly deliver with an NSAID and aglycosamineglycan like glucosamine. In experiments showing transport ofglucosamine and glucosamine mutual prodrugs across shed snake skin,glucosamine HCl was not transported across the shed snakeskin, whereasN-acetylglucosamine (NAG) was highly transported, even though NAG haswhat is considered a poor partition coefficient of about 0.017 inoctanol/water.

Compound 6 has a partition coefficient (calculated in silico log P) of6.45, and compound 9 has a partition coefficient (calculated in silicolog P) of 3.81, indicating an expectation of greater lipid solubilityand greater possibilities for transport to occur. It has been shown thatboth ibuprofen and ketoprofen are transported across shed snakeskin(U.S. Pat. No. 6,368,618 B1; Phar acta Helv 1996, August, 7(3):205-212;boll Chim. Farm. 2000 March-April, 139(2):67-72); both ketoprofen andibuprofen are lipid soluble NSAIDS. It is expected that theNSAID-glucosaminglycan mutual prodrug will also be transported acrossshed snakesin as a model for the human epidermis; it is also expectedthat an NSAID ester prodrug will exhibit similar transport properties.

indicating greater possibilities for transport of the mutual prodrug tooccur.

Example 3 Transport of Glucosamines and Glucosamine Salts Across Skin

Oral administration of glucosamine, its derivatives and analogs, forexample N-acetyl-D-glucosamine, are affected by the liver's first-passmetabolism (Setnikar et al., Arzneimittel-Forschung, 36(4):729-35(1986); Du et al., Biopharmaceutics & Drug Disposition, 25(3):109-116(2004)). However, a more recent report indicates that these agents maybe metabolized mostly in the gut rather than solely by the liver(Aghazadeh-Habashi et al., Journal of Pharmacy & PharmaceuticalSciences, 5(2):181-184 (2002)). Few pharmacokinetic literature reportsexist on the disposition of these agents in articular cartilage(Setnikar et. al., Arzneimittel-Forschung, 43(10):1109-1113 (1993) andArzneimittel-Forschung, 51(9):699-725 (2001)) have reported on thepharmacokinetic properties of glucosamine in dogs and man. It isestimated that approximately 87% of the original glucosamine oral doseis absorbed and excreted; <13% is widely distributed in the body; and<1% reaches osteoarthritic joints. Chondroitin is known to degrade intoits basic dissaccaride components within the gut prior to furthermetabolism (Lamari et al., Biomed. Chromatogr., 16:95-102 (2002)).Although only a small fraction of glucosamine reaches the articularcartilage target site, it is reported to exhibit a high potency; andtogether glucosamine and chondroitin therapy demonstrate therapeuticefficacies over time, (McAlindon et al., JAMA, 283:1469-1475 (2000)).

Initial data shows transport of certain topically delivered glucosaminecompositions of the present invention (esters of glycosaminoglycan) ascompared with glucosamine-containing creams of the type currentlyavailable. These creams typically include glucosamine salts (HCl orsulfate), which are monovalent (uncharged) chemical entities that do notcross and/or penetrate the skin unless an electrical charge is applied.

The compositions analyzed included glucosamine HCl, N-acetylglucosamine, and glucosamine-pentaacetyl as described below, in asolution of DMSO:

Glucosamine HCl

Molecular Formula=C₁₄H₂₂ClNO₉

Formula Weight=383.7785

Composition=C(43.81%) H(5.78%) Cl(9.24%) N(3.65%) O(37.52%)

Molar Refractivity=Not available

Molar Volume=Not available

Parachor=Not available

Index of Refraction=Not available

Surface Tension=Not available

Density=Not available

Dilectric Constant=Not available

Polarizability=Not available

Monoisotopic Mass=383.098312 Da

Nominal Mass=383 Da

Average Mass=383.783195 Da

N-acetyl Glucosamine

Molecular Formula=C₁₄H₂₁NO₉

Formula Weight=347.3179

Composition=C(48.41%) H(6.09%) N(4.03%) O(41.46%)

Molar Refractivity=77.50±0.4 centimeters (cm)³

Molar Volume=266.2±5.0 cm³

Parachor=703.1±6.0 cm³

Index of Refraction=1.493±0.03

Surface Tension=48.6±5.0 dyne/cm

Density=1.30±0.1 gram/cm³

Dilectric Constant=Not available

Polarizability=30.72±0.5×10⁻²⁴ cm³

Monoisotopic Mass=347.121634 Da

Nominal Mass=347 Da

Average Mass=347.32248 Da

Glucosamine Pentaacetyl

Molecular Formula=C₁₆H₂₃NO₁₀

Formula Weight=389.3546

Composition=C(49.36%) H(5.95%) N(3.60%) O(41.09%)

Molar Refractivity=86.91±0.4 cm³

Molar Volume=299.4±5.0 cm³

Parachor=787.0±6.0 cm³

Index of Refraction=1.492±0.3

Surface Tension=47.6±5.0 dyne/cm

Density=1.30±0.1 gram/cm³

Dilectric Constant=Not available

Polarizability=34.45±0.5×10⁻²⁴ cm³

Monoisotopic Mass=389.132199 Da

Nominal Mass=389 Da

Average Mass=389.359811 Da

In vitro transport (diffusion) was evaluated by Franz cell diffusionexperiments using shed snakeskin. Shed snakeskin is widely recognized asa sufficient model membrane to human skin for preliminary permeabilitystudies due to the similarity in its composition to the human stratumcorneum.

The shed snakeskin was hydrated in 0.1% aqueous sodium azide solutionfor 48 hours at room temperature. The skins were mounted to three Franzreceptor cells filled with 0.1 M pH7 phosphate buffer. The receptorsolution was maintained at 37° C. and stirred with a magnetic stirrer.The donor cells were clamped to each receptor cell, with the skinmounted between the receptor and donor cells, and the donor cells werefilled with 100 milligrams (mg) each of glucosamine HCl (e.g., cell 1),N-acetyl glucosamine (e.g., cell 2), and glucosamine pentaacetyl (e.g.,cell 3) in a 1 milliliter (ml) DMSO solution. The skin surface exposedto diffusion was 2.54 cm² (1.8 cm diameter) and the receptor cell volumewas 6 cm³. The system was allowed to equilibrate for two hours beforesamples were taken.

Twenty microliter (μl) samples of receptor solution were taken at 5, 10,20, 40, 80, 160, and 240 minute intervals and replaced with freshbuffer. A ten μl aliquot of each sample was analyzed by high performanceliquid chromatography with pulsed electrochemical detection.

As shown in FIG. 12, there was no observable diffusion and/or transportacross the skin membrane of the glucosamine HCl salt composition. Thecompositions of esters of glycosaminoglycan (N-acetyl glucosamine andglucosamine-pentaacetyl) showed immediate and constant diffusion and/ortransport from 5 minutes to 240. Each receptor cell volume was analyzed,revealing over 50% transport of esters (e.g., of the 100 mg of theesters in the delivery phase, over 50 mg of the esters were deliveredover time.

Example 4 Transdermal Permeability of N-acetyl-D-glucosamine

As an objective of this research was to evaluate transdermalpermeability of glucosamines, esters, and derivatives thereof, to assessthe feasibility of pursuing a percutaneous formulation for localtherapy, NAG was selected for analysis because it is an activemetabolite and prodrug of glucosamine; and owing to its commercialavailability, relatively low cost and stability. It possesses thefollowing physical and chemical characteristics making it a reasonablecandidate for transdermal delivery and percutaneous absorption: a) highpotency, b) reasonably lipid soluble, c) low molecular weight, d) uniquebiochemical pathway with active transport from blood into articularcartilage. (Milewski, Biochimica et Biophysica Acta, 1597:173-192(2002)). Furthermore, exogenous glucosamine is understood to promoteglycosaminoglycan synthesis toward the production of proteoglycans byavoiding the rate-limiting steps of its conversion from glucose toglucosamine, and ultimately to N-acetyl-D-glucosamine by glutamine(fructose-6-phosphate amidotransferase) (McClain et al., Diabetes,45:1003-1009 (1996)). Glucose and glucosamine are substrates ofglucokinase (Singh et al., Diabetes, 50:2355-2362 (2001)). Thephosphorylated glucosamine product, glucosamine-6-phosphate, inhibitsgluokinase and alters both glucose and subsequent glucosamine metabolism(Van Schaftigen et al., Biochem. J., 308:23-29 (1995)). Miwa et al.reported that glucokinase has a low affinity for NAG (Enzyme Protein,48:135-142 (1994)). Thus, NAG kinase mediates the phosphorylation of NAGto produce NAG-6-phosphate that does not affect glucokinase activity(Miwa et al., Enzyme Protein, 46:135-142 (1994)), allowing glucose andglucosamine to proceed through metabolism unrestricted. Thus, thebiosynthesis of glycosaminoglycans from this perspective was believed tobe better promoted, in certain embodiments and for certain specificuses, by the use of NAG or some other rate-limiting glucosamine analograther than by glucosamine (Shikhman et al., J. Immunol., 166:5155-5160(2001)), although the use of glucosamine is not precluded.

Permeability was evaluated by employing NAG suspensions of various knownmembrane transport enhancing reagents, ethanol, oleic acid, isopropylmyristate, isopropyl palmitate; NAG solutions of water and phosphatebuffer; and NAG saturated dimethylsulfoxide (DMSO) solution.

Glucosamine and chondroitin salts are charged, highly polar, aqueoussoluble, and poor candidates for transdermal absorption. Currently,there are topical products containing these ingredients as saltsmarketed nutraceuticals for the treatment of osteoarthritis, whichcontains ingredients whose effects may be mistaken in the short term asbeing therapeutic NAG an acetylated glucosamine metabolite is less polarand neutral appears to be a more likely candidate for transdermaldelivery and percutaneous absorption.

Glucosamine is metabolized to NAG via the hexosamine pathway;glucosamine or galactosamine, plus a uronic acid, is incorporated as adisaccharide unit into all macromolecules requiring amino sugars such askeratan, dermatan, chondroitin, hyluronates, and heparin, to produceglycosaminoglycans (GAGs). GAGs are highly negatively charged molecules,with an extended conformation, and demonstrate high viscosity and lowcompressibility ideal as a lubricating fluid for anatomical joints. Themajority of GAGs in the body are linked to core proteins, to formproteoglycans or mucopolysaccarides, which are basic components of skin,tissue, and cartilage. (Merrick et al., J. Bio. Chem. 5:235 (1960);Milewski, “Glucosamine-6-phophate synthase,” Biochimica et BiophysicaActa, 1597:173-192 (2002).

Materials and Methods

Chemicals

NAG of 99.9+% purity was purchased from MP Biomedical (Aurora, Ohio).All enhancer reagents purchased for this study were at 99.9+% purity.All other reagents were of analytical grade and used without furtherpurification.

Analysis

NAG analysis was carried out using high-performance anion exchangechromatography with pulsed amperometric detection (HPAE-PAD) on a DionexDX-500 HPLC system (Dionex, Sunnyvale, Calif.) that included a GP40gradient pump, ED40 Electrochemical detector, AS3500 autosampler and aPEAKNET Chromatography Workstation, (“Optimal Settings for PulsedAmperometric Detection of Carbohydrates Using the Dionex ED40Electrochemical Detector,” Technical Note 21, Dionex Corp., Sunnyvale,Calif., USA.; Clarke et al., Anal Chem, 71:2774-2781 (1999); Campo etal., J. Chrom. B, 765:151-160 (2001); LaCourse, W. R. PulsedElectrochemical Detection in High-Performance Liquid Chromatography,John Wiley & Sons Inc. (1997)). The HPAE-PAD was equipped with aCARBOPAC PA20 (3×150 mm), analytical anion-exchange column (Dionex,Sunnyvale, Calif.) for the rapid, high-resolution separation ofmonosaccharides and disaccharides, using pulsed amperometric detection,a CARBOPAC PA20 analytical guard column (3×30 mm) (Dionex, Sunnyvale,Calif.), and a carbonate trap column (25×15 mm) (Dionex, Sunnyvale,Calif.). Mobile phase (A) was degassed and prepared with deionizedwater. The mobile phase (B) included 0.02 N NaOH prepared with deionizedwater and filtered with 0.45 micrometer (μm) filters in a solventfiltration apparatus (Waters-Millipore, Milford, Mass., USA) that wasdegassed under vacuum. The mobile phase system was run at a gradientconcentration of 16 mM NaOH at a flow rate of 0.5 milliliters per minute(ml/min). A standard calibration curve of NAG (FIG. 13) was obtainedwith linear regression and value of R²=0.9936. Each sample set was runwith external standards. The sample concentration values were obtainedvia the PEAKNET software. These values were compared with to thoseobtained by calculations of the peak area and peak height observe asfunctions of the standard curve's linear regression equation. Theinstrument sensitivity was approximately 10⁻⁴ units.

Solubility Measurements

An excess amount of NAG (pKa 6.73) was placed in separate vialscontaining 10 ml deionized water, 10 ml n-hexane and 10 ml phosphatebuffer (pH 6, 6.73, and 7.4; 1 M) and stirred at 37° C. for 24 hours.The solutions were centrifuged for 5 min at 9000 rev/min and thesupernatant filtered with cellulose acetate membrane filters (0.45 μmpore size) (Dionex, Sunnyvale Calif.). The NAG concentration in eachfiltrate was determined by HPAE-PAD after the appropriate dilution.

Determination of Partition Coefficients

The oil/water partition coefficient for NAG was determined usingn-hexane/phosphate buffer (pH 5.5 6, 6.73, and 7.4, 0.1 M) andn-hexane/water (Bemacki et al., J. Supramolecular Structure, 7:235-250(1977)). In each case 5 ml of n-hexane was mixed with aqueous solutionscontaining NAG and shaken at 37° C. for 24 hours. The mixture wasafterwards centrifuged and the organic and aqueous phases separated. TheNAG concentration in the filtrates was determined by HPAE-PAD after theappropriate dilution.

In-Vitro Membrane Permeation

Shed snakeskins were used as a model membrane for permeation studiesusing the NAG suspensions in known membrane permeation enhancers;ethanol, oleic acid, isopropyl myristate, and isopropyl palmitate;saturated solutions of NAG in water and in phosphate buffer; as well asin a saturated DMSO solution and phosphate buffer (pH 5.5) containingethanol at 2%, 5%, 10%, 25%, and 50% solutions.

The skins were hydrated in 0.1% aqueous sodium azide solution at roomtemperature for 48 hrs. Franz-cell diffusions experiments were carriedout. In general the receptor cell was filled with 7.4 pH 0.1 M phosphatebuffer and the donor cell filled with a solution or suspension. For thephosphate buffer (pH 5.5) containing ethanol at 2%, 5%, 10%, 25%, and50% solutions in the donor phase, the receptor phase consisted ofphosphate buffer (pH 5.5, 0.1 M). The receptor solution was maintainedat 37° C. and stirred with a magnetic stirrer.

The snake skins were mounted between the receptor and donor cells. Thesurface exposed to diffusion was 2.54 cm² (diameter 1.8 cm) and thereceptor cell volume was 6 cm³. The donor cell was covered with plasticfilm. The system was allowed to equilibrate at 37° C. for two hoursbefore each experiment. To the donor cells, 5 ml of the NAG-enhancersuspension or solution was added. Samples were taken at intervals over a24-hour period, 200-μl samples of receptor solutions were taken andreplaced with fresh buffer; experiments were conducted in triplicate.The amounts of NAG that permeated through the snakeskin were determinedby HPAE-PAD.

Data Treatment

Steady state flux (J_(ss)) for NAG (mg/cm²/h) was calculated from itsincreasing amount in the receptor medium (Bach et al., Eur. J. Pharm.Biopharm, 46:1-13 (1998)). NAG's permeability coefficient (k_(p)) incm/h was calculated from known physiochemical parameters, (Hadgraft etal., “Feasibility Assessment in Topical and Transdermal Delivery:Mathematical Models and In Vitro Studies,” in Transdermal Drug Delivery.2^(nd) Ed. Marcel Dekker, Inc., pages 1-23 (2003)). Lag time (t_(lag))was determined graphically from the cumulative amount of drug releasedper unit area (mg/cm²) versus time plots. A square root of time(t^(1/2)) versus cumulative amount of drug released per unit area(mg/cm²) was obtained to monitor NAG in vitro release rate (mg/cm²),(Guidance for Industry: SUPAC-SS Semisolid Dosage Forms. Scale-up andPostapproval Changes Chemistry, Manufacturing, and Control; In VitroRelease Testing and In Vivo Bioequivalence Documentation. US Departmentof Health and Human Services, Food and Drug Administration, Center for.Drug Evaluation and Research, May 1997).

Results

Initial permeability investigations were carried out using shedsnakeskin as a model membrane to human skin; a widely recognized andsufficient model for preliminary studies due to its similarity incomposition to the human stratum corneum (Itoh et al., “Use of ShedSnake Skin as a Model Membrane for In Vitro Percutaneous PenetrationStudies: Comparison with Human Skin,” Pharm. Res., 7:1042-1047 (1990)).Negligible NAG transport was observed from suspensions of membranepermeability enhancers; ethanol, oleic acid, isopropyl myristate andisopropyl palmitate. No permeation was observed from the aqueoussolutions of NAG in water or phosphate buffer solutions (pH 5.5, 6.0,6.73, and 7.4; 0.1 M). As a qualitative correlation to a selection ofNAG's partition coefficients shown in Table 1, permeation of NAG fromthe aforementioned membrane penetration enhancer suspensions or aqueoussolutions was expected.

TABLE 1 Experimentally determined partition coefficients for N-acetyl-D-glucosamine (NAG) (pKa 6.73) and permeability coefficient (k_(p))n-hexane/ n-hexane/ n-hexane/ n-hexane/ pH 5.5 pH 6.0 pH 6.73 pH 7.4Octanol/ buffer buffer buffer buffer water¹⁵ k_(p)(cm/hr) 0.252 0.1940.092 0.091 0.017 0.731 ¹⁵Bernacki et al., J. Supramolecular Structure,7: 235-250 (1997)

DMSO was chosen for evaluation as a benchmark permeation enhancer due toits physical properties and well-documented enhancement properties(Franz et al., “Dimethyl sulfoxide,” in Percutaneous Enhancers. Ed.Smith, E. W., Maibach, H. I, CRC Press, Inc., pages 112-127 (1995)).Enhancers are reported to disrupt intercellular lipids of the stratumcorneum, by increasing a drug's partitioning into the stratum corneumwith a concomitant increase in drug permeation through the intercellularjunctions via percutaneous absorption. (Barry, J. Control. Rel.,15:237-248 (1991); Williams et al., Crit. Rev. Ther. Drug Carrier Syst.,9:305-353 (1992); Sinha et al., Drug Dev. Ind. Pharm., 26:1131-1140(2000)). From the plot containing cumulative NAG concentration per unitarea (mg/cm²) versus time^(1/2) (hour^(1/2)) NAG's in vitro release ratewas shown to be 73.48 μg/cm² (FIG. 13) with high linearity in transportthus exhibiting no lag time, as indicated in Table 2. The assumptiontaken is that NAG's high polarity and its low permeation coefficient, asshown in Table 3, contributes to its inability to be transportedefficiently by means of single permeation enhancer or in aqueoussolution.

Table 2. Physiochemical data obtained for the permeation ofN-acetylglucosamine (NAG) through shed snake's skin via a saturateddimethyl sulfoxide (DMSO) solution in the donor phase and pH 7.4phosphate buffer in receptor phase.

TABLE 2 Physiochemical data obtained for the permeation ofN-acetylglucosamine (NAG) through shed snake's skin via a saturateddimethyl sulfoxide (DMSO) solution in the donor phase and pH 7.4phosphate buffer in receptor phase. Parameter J_(ss) (mg/cm²/h) 73.48t_(lag) (h) In Vitro release rate (mg/cm²) 186.64 R² 0.9736

TABLE 3 Physicochemical data obtained for the permeation ofN-acetylglucosamine (NAG) through shed snake's skin via phosphate buffer(pH 5.5) containing ethanol at 2%, 5%, 10%, 25%, and 50% solutions inthe donor phase and pH 5.5 phosphate buffer in the receptor phase. (%Ethanol) Parameter 2 5 10 25 50 J_(ss) 112.61 119.53 211.61 205.93 77.96(mg/cm²/h) In Vitro 286.03 303.61 537.49 523.06 198.02 release rate(μg/cm²) t_(lag) (h) R² 0.9778 0.8751 0.7966 0.9924 .9836

The study shows that DMSO allows NAG to be transported immediately andcontinuously with a linear concentration increase over time, asevidenced in FIG. 13, which shows the effect of DMSO on the cumulativepermeation of NAG at 37.5° C. through shed snake skin (cumulativeconcentration vs time^(1/2), each point representing the mean+/−standard deviation, n=3).

This study also incorporated NAG into an ethanol/buffer solution atvarious concentrations. Ethanol as an enhancer is known to promote thetransdermal penetration and percutaneous absorption of many drugs,(Berner et al., “Alcohols, Percutaneous Penetration Enhancers,” inAlcohols. Eds. Smith, E. W. Maibach, H. I., CRC Press, Boca Raton, Fla.,pages 45-60 (1995)). The oil/water partition coefficient increases withthe decrease in pH of the buffer solution, shown in Table 1. NAG'stransdermal transport was not observed from phosphate buffer or ethanolwhere it is highly soluble and insignificantly soluble respectively. Itspermeation was observed in sink conditions from the phosphate buffer (pH5.5) containing ethanol at 2%, 5%, 10%, 25%, and 50%, as reported inFIG. 14, showing accumulation of NAG through shed snake skin in Franztype receptor cells from phosphate buffer (pH 5.5) after 24 hours. Thecumulative concentration of the solutions containing 5%, 10%, and 25%ethanol are very similar after 24 hrs, whereas the 2% and 50% ethanol inbuffer solutions delivery significantly less NAG (FIG. 15, showing theeffect of ethanol concentration cumulative permeation of NAG at 37.5° C.through shed snake skin, cumulative concentration versus time^(1/2)).Beyond 50% ethanol concentration in buffer, the NAG precipitated. Theflux values for 10% and 25% ethanol concentration are similar, whereasthe 5% is half that of both. Graphically, each has a slightly differentlinear permeation profile up to the approximate experimental mid-point.At the conclusion of the 24-hour endpoint each solution had deliveredsimilar amounts of NAG.

These results overall suggest that thermodynamic and solubility effectsaffect the permeation of NAG in correlation to use of DMSO at 100%(Kurihara-Bergstrom et al., J. Inv. Derma., 89:274-280 (1987)) and thevarying concentrations of ethanol in buffer solutions. NAG's in vitroflux and release rates from ethanol in buffer solutions overall exceededthose permeation values obtained from DMSO. This shows that 5-25%concentration of ethanol as an enhancer in delivery vehicles to be auseful starting point towards the formulation of a transdermallydelivered/percutaneously absorbed NAG composition.

The current study also shows DMSO to be a skin penetration enhancer forNAG. DMSO is generally used in veterinary drug delivery, (Magnusson etal., Adv. Drug Del. Rev., 50:205-227 (2001)). The use of DMSO in NAGformulation may be useful for localized osteoarthritis treatment inanimals, since DMSO is not an FDA approved excipient for human use intopical/transdermally delivered pharmaceutical products. Furthermore,unpublished preliminary results in our laboratory show that NAG may haveadequate permeation from other drug delivery vehicles. It is anticipatedthat further in vitro studies will determine other NAG formulations willeffectively demonstrate transdermal delivery towards percutaneousabsorption.

Example 5 Permeation of N-acetyl Glucosamine (NAG) in PluronicOrgano-Gel Formulations Across Shed Snakeskin

The objective of this study was to further evaluate the permeation ofNAG in pluronic-organo-gel formulations across shed snake's skin. Thesepluronic gel formulations each contained lecithin-isopropyl palmitateand/or lecithin-vitamin E component as the enhancer and organic phase.Isopropyl palmitate is a well-studied enhancer. Sparse literaturereports concerning the permeation enhancement effects of vitamin E andsoy lecithin are available other than patents and patent applicationsdescribing various dosage forms.

For these studies, NAG was chosen as the compound of choice, even thoughit has a octanol-water partition coefficient of 0.017 (pH 7.4) (Bernackiet al., J. Supramolecular Structure, 7:235-250 (1977)). Mahjour andco-workers (Journal of Controlled Release, 14(3):243-52 (1990)) studiedthe effect of lecithins on in vitro skin permeability with several drugswith various NAG's octanol-water partition coefficients. They found thatsoy lecithins improved the permeability of all the drugs includingprocaterol and oxymorphone which both have a comparatively lowoctanol-water partition coefficient of −0.37 (pH 7.7) and 0.0 (pH 7.4)respectively. The most impressive finding was that the soy lecithinsenhancement effect was the highest for procaterol, which has the lowestoctanol-water partition coefficient of the drugs studied (Mahjour etal., Journal of Controlled Release, 14(3):243-52 (1990)). As for vitaminE, it is postulated to act as a permeation enhancer by intercalatingwithin the lipid bilayer of the stratum corneum, which alters themembrane's permeability (Trivedi et al., European Journal ofPharmaceutical Sciences, 3(4):241-243 (1995)).

Vitamin E has a two-fold effect within biological membranes as anantioxidant and membrane stabilizer. Structurally, vitamin E's chromanolring's hydroxyl group is believed to situate in the polar headenvironment of the phospholipid membrane, while the phytyl chainintercalates with the lipid acyl chains. Trivedi and co-workers observedvitamin E's permeation enhancement (European Journal of PharmaceuticalSciences, 3(4):241-243 (1995)). Their conclusion was that overallimprovement in the permeability of the stratum corneum is moderate. Thisis a result from the limited insertion of vitamin E within theceramide-rich bilayer structure. Thus their final concluding statementwas that overall vitamin E enhancement effect “ . . . will not betremendous but discernible nonetheless (Trivedi et al., European Journalof Pharmaceutical Sciences, 3(4):241-243 (1995)).

Experimental

Materials

NAG was purchased from MP Biomedicals, Inc (Aurora, Ohio). Poloxamer 407(PLURONIC F127; Polyethylene-Polypropylene Glycol), soy lecithin andisopropyl palmitate were purchased from Spectrum Chemical Mfg. Corp.(Gardena, Calif.). Vitamin E (mixed tocopherol complex) was purchasedfrom Solgar Vitamin and Herb (Leonia, N.J.). Water used for thepreparation was double distilled and deionized by a Milliporepurification system (Continental Water Systems Corp., El Paso, Tex.).

Vehicle Preparation

Twenty percent poloxamer 407 (PLURONIC F127; Polyethylene-PolypropyleneGlycol) gels were made by standard methods e.g. dissolving the poloxamerinto cold water under refrigeration for 24 hours to produce the pluronicgel phase. NAG was incorporated into the pluronic gel phase. The finalformulations were prepared via the emulsification of the select organicphase with the pluronic gel phase using luer-lock connected syringes.

In-Vitro Membrane Permeation Studies

Shed snakeskins were used as the model membrane for these permeationstudies using the NAG semi-solid gel formulations. The skins werehydrated in 0.1% aqueous sodium azide solution at room temperature for48 hrs. Franz-cell diffusions experiments were carried out. In generalthe receptor cell was filled with a 7.4 pH 0.1 M phosphate buffer. Thereceptor solution was maintained at 37° C. and stirred with a magneticstirrer. The snakeskins were mounted between the receptor and donorcells. The surface exposed to diffusion was 2.54 cm² (diameter 1.8 cm)and the receptor cell volume was 6 cm³. The donor cell was covered withplastic film. The system was allowed to equilibrate at 37° C. for twohours before each experiment. The donor cells were filled with a 5 ml ofthe semisolid NAG pluronic-organo-gel formulation. Samples (N=3) weretaken at intervals over a 48-hour period, 200-μl samples of receptorsolutions were taken and replaced with fresh buffer; experiments wereconducted in triplicate. The amounts of NAG that permeated through thesnakeskin were determined by HPAE-PAD.

HPAE-PAD (HPLC) Analysis

NAG analysis was carried out at the University of Georgia, Center forComplex Carbohydrate Research Center. High-performance anion exchangechromatography with pulsed amperometric detection (HPAE-PAD); Dionex,Sunnyvale, Calif. USA); on a Dionex DX-500 HPLC system consisting of aP40 gradient pump, ED40 Electrochemical detector, AS3500 autosampler andPeakNet Chromatography Workstation was utilized. The HPAE-PAD wasequipped with CARBOPAC PA20 (3×150 mm), analytical anion-exchange columnfor the rapid, high-resolution separation of monosaccharides anddisaccharides, using pulsed amperometric detection and a CARBOPAC PA20analytical guard column (3×30 mm) and a carbonate trap column (25×15mm). Mobile phase (A) was degassed and prepared with deionized water.The mobile phase (B) consisted of 0.02 N NaOH prepared with deionizedwater and filtered with 0.45 μm filters in a solvent filtrationapparatus (Waters-Millipore, Milford, Mass., USA) that was degassedunder vacuum. The mobile phase system was run at a gradientconcentration of 16 mM NaOH at a flow rate of 0.5 ml/min. A standardcalibration curve of NAG (FIG. 16, showing the effect of soylecithin-vitamin E on NAG permeation across shed snake skin, cumulativeconcentration (n=3) per unit area+/− standard deviation as a function oftime^(1/2)) was obtained with linear regression and value of R²=0.9936.Each sample set was run with external standards. The sampleconcentration values were obtained via the PEAKNET software. Thesevalues were compared with to those obtained by calculations of the peakarea and peak height observe as functions of the standard curve's linearregression equation. The instrument sensitivity was approximately 10⁻⁴units.

Data Analysis

Steady state flux (J_(ss)) for NAG (mg/cm²/h) was calculated from itsincreasing amount in the receptor medium (Bach et al., Eur. J. Pharm.and Biopharm., 46:1-13 (1998)). NAG's permeability coefficient (k_(p))in cm/h was calculated from known physiochemical parameters (Hadgraft etal., “Feasibility Assessment in Topical and Transdermal Delivery:Mathematical Models and In Vitro Studies,” in Transdermal Drug Delivery,2^(nd) Ed. Marcel Dekker, Inc., pages 1-23 (2003)). Lag time (t_(lag))was determined graphically from the cumulative amount of drug releasedper unit area (mg/cm²) versus time (h) plots. A square root of time(t^(1/2)) versus cumulative amount of drug released per unit area(mg/cm²) was obtained to monitor NAG in vitro release rate (mg/cm²)(Guidance for Industry: SUPAC-SS Semisolid Dosage Forms. Scale-up andPostapproval Changes Chemistry, Manufacturing, and Control; In VitroRelease Testing and In Vivo Bioequivalence Documentation. US Departmentof Health and Human Services, Food and Drug Administration, May 1997).

Results

Initially NAG transport was observed from lecithin-isopropyl palmitate,vitamin E, and lecithin-vitamin E (1:1) suspensions (100 mg/ml).Negligible (below the limit of detection) transport was observed fromall of these suspensions, excluding the permeation of NAG from thelecithin-vitamin mixed E (1:1) suspension shown in FIG. 17, showingphysicochemical data obtained for the permeation of NAG in pluronicgel-organic phase vehicles through shed snake skin, cumulativeconcentration (n=3) per unit area+/− standard deviation as a function oftime^(1/2) (IA: 1:1 w/w of pluronic gel:organic phase IA(lecithin:vitamin E (1:1 w/w)); IIA: 4:1 w/w of pluronic gel:organicphase IIA (lecithin:vitamin E (1:1 w/w)); IIIA: 0.93:0.07 w/w ofpluronic gel:organic phase IIIA (lecithin:vitamin E (0.93:0.07 w/w));IB: 1:1 w/w of pluronic gel:organic phase IB (lecithin:isopropylpalmitate (1:1 w/w)); IIB: 1:1 w/w of pluronic gel:organic phase IIB(lecithin:isopropyl palmitate (1:1 w/w)); IIIB: 0.93:0.07 w/w ofpluronic gel:organic phase IB (lecithin:isopropyl palmitate (1:1 w/w)).

Relatively, linear transport was observed from the lecithin-vitaminmixed E (1:1) suspension. NAG's release rate=13.71 μg/cm², steady stateflux J_(ss)=4.23 μg/cm²/h and its permeability coefficientk_(p)=5.012×10⁻³ (cm/h) have been recorded (Table 4).

TABLE 4 Physicochemical data obtained for the permeation ofN-acetylglucosamine (NAG) in a lecithin-vitamin pluronic-organic phasevehicle through shed snakeskin. Release Vehicle J_(ss) (mg/cm²/h) rate(mg/cm²) k_(p) (cm/h) t_(lag) (h²) Lecithin- 4.23 g/cm²/h 13.71 μg/cm²5.012 × 10⁻³ 0 Vitamin E (1:1)

For the pluronic organo-gels, each vehicle was composed of a pluronicgel and organic phase, as shown in Table 5, along with its respectivephysicochemical data obtained for the permeation of N-acetylglucosamine(NAG). Each NAG vehicle exhibited lag time, as reported in Table 5. Thecumulative concentration versus time plot to obtain t_(lag) is notshown. As exhibited in FIG. 17, the cumulative concentration per unitarea versus the square root of time plot shows a comparison of all thevehicles. Overall graphically, the pluronic gel formulations containingorganic phase mix of lecithin and isopropyl palmitate out-performed thepluronic gel containing lecithin and vitamin E organic phase vehicles.Formulation III A, which contained the pluronic gel to lecithin-vitaminE mix at a 0.93:0.7 exhibited the best release rate, steady state fluxand permeability coefficient values (Table 5).

The data shows that as the organic enhancer phase is decreased, NAGpermeation increases for both formulations III A and III B. However, therelease rates of formulations II B and III B are comparable.Correspondingly, in experiments not reported here, NAG was nottransported in aqueous only vehicles.

TABLE 5 Physicochemical data obtained for the permeation of NAG inpluronic-organic phase vehicles through shed snakeskin. Graphical J_(ss)Release rate k_(p) t_(lag) Vehicle Label Organic Phase (mg/cm²/h)(mg/cm²) (cm/h) (h) Pluronic Gel: Organic Phase I A I A Lecithin:Vitamin E (1:1 w/w) 1.01 6.88 3.2 × 10⁻³ 6.45 (1:1) Pluronic Gel:Organic Phase II A II A Lecithin: Vitamin E (1:1 w/w) 1.06 8.94 3.1 ×10⁻³ 3.98 (4:1) Pluronic Gel: Organic Phase III A III A Lecithin:Vitamin E (1:1 w/w) 10.3 74.9 3.2 × 10⁻¹ 0.85 (0.93:0.7) Pluronic Gel:Organic Phase I B I B Lecithin: Isopropyl palmitate E 2.16 14.6 3.1 ×10⁻⁴ 5.33 (1:1) (1:1 w/w) Pluronic Gel: Organic Phase II B II BLecithin: Isopropyl palmitate E 6.98 38.5 2.5 × 10⁻⁴ 6.98 (4:1) (1:1 w/wPluronic Gel: Organic Phase III B III B Lecithin: Isopropyl palmitate E4.46 43.1 6.8 × 10⁻⁴ 2.49 (0.93:0.7) (1:1 w/w

The complete disclosures of all patents, patent applications,provisional patent applications, publications, and electronicallyavailable material cited herein are incorporated by reference. Theforegoing detailed description and examples have been provided forclarity of understanding only. No unnecessary limitations are to beunderstood therefrom. The invention is not limited to the exact detailsshown and described; many variations will be apparent to one skilled inthe art and are intended to be included within the invention defined bythe claims.

1. A pharmaceutical composition comprising a therapeutically effectiveamount of a compound having formula II:

wherein R¹, R², R³, R⁴ and R⁵ are each independently H or an organicgroup; and a pharmaceutically acceptable carrier; wherein thecomposition is formulated for transdermal application and the permeationcoefficient k_(p) of the compound of formula II in shed snake skin is atleast about 2.5×10⁻⁴ cm/h.
 2. The pharmaceutical composition of claim 1further comprising at least one anti-inflammatory agent.
 3. Thepharmaceutical composition of claim 1 wherein the carrier is selectedfrom the group consisting of an ointment, a gel, a cream, a solution, alotion, a suspension, a microemulsion, an emulsion, a liposome, or atransdermal patch.
 4. A pharmaceutical composition comprising atherapeutically effective amount of a compound having formula II:

wherein R¹, R², R³ and R⁴ are each H, and R⁵ is an acetyl group; and apharmaceutically acceptable carrier; wherein the composition isformulated for transdermal application and the permeation coefficientk_(p) of the compound of formula II in shed snake skin is at least about2.5×10⁻⁴ cm/h.
 5. The pharmaceutical composition of claim 4 furthercomprising an anti-inflammatory agent.
 6. The pharmaceutical compositionof claim 1 further comprising one or more additional components selectedfrom the group consisting of an antimicrobial agent, a gelling agent, anemulsifying agent, a stiffening agent, a skin healing agent, anemollient, a surfactant, a solvent, a lubricant, a wax, a humectant, askin penetration enhancer, an antioxidant, and combinations thereof. 7.The pharmaceutical composition of claim 6 wherein the skin penetrationenhancer is selected from the group consisting of dimethyl sulfoxide,ethanol, isopropyl palmitate, isopropyl myristate, cetyl alcohol,stearyl alcohol, cetostearyl alcohol, polyethylene glycol, urea,dimethyl acetamide, sodium lauryl sulfate, Spans, Tweens, terpenes,azone, acetone, oleic acid, and combinations thereof.
 8. Thepharmaceutical composition of claim 4 further comprising one or moreadditional components selected from the group consisting of anantimicrobial agent, a gelling agent, an emulsifying agent, a stiffeningagent, a skin healing agent, an emollient, a surfactant, a solvent, alubricant, a wax, a humectant, a skin penetration enhancer, anantioxidant, and combinations thereof.
 9. The pharmaceutical compositionof claim 8 wherein the skin penetration enhancer is selected from thegroup consisting of dimethyl sulfoxide, ethanol, isopropyl palmitate,isopropyl myristate, cetyl alcohol, stearyl alcohol, cetostearylalcohol, polyethylene glycol, urea, dimethyl acetamide, sodium laurylsulfate, Spans, Tweens, terpenes, azone, acetone, oleic acid, andcombinations thereof.